Insect inhibitory bacillus thuringiensis proteins, fusions, and methods of use therefor

ABSTRACT

Novel insect inhibitory proteins are disclosed comprising two different components, both of which are required for biological activity. Various methods of linking both components together, so that a single protein provides insect inhibitory activity, are disclosed. Also disclosed are novel  Bacillus thuringiensis  nucleic acid sequences encoding Coleopteran-inhibitory crystal proteins, designated tIC100 (29-kDa) and tlC101 (14-kDa). Also disclosed are methods of making and using nucleic acid sequences in the development of the transgenic plant cells containing the novel nucleic acid sequences disclosed herein.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S.Provisional Application No. 60/232,099, filed Sep. 12, 2000.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the field of molecularbiology. More particularly, the present invention concerns a new classof insect inhibitory proteins comprising two different components, bothof which are required for biological activity. The present inventionconcerns the construction of coleopteran-inhibitory crystal proteins, inparticular CryET33/CryET34 and tIC100/tIC101 from Bacillusthuringiensis. Various methods of linking the proteins together, so thata single protein provides insect inhibitory activity, are disclosed. Theuse of nucleic acid sequences as diagnostic probes and templates forprotein synthesis, and the use of polypeptides, fusion proteins,antibodies, and peptide fragments in various insect inhibitory,immunological, and diagnostic applications are also disclosed, as aremethods of making and using nucleic acid sequences in the development oftransgenic plant cells containing the nucleic acid sequences disclosedherein.

[0004] 2. Description of the Related Art

[0005] Environmentally-sensitive methods for controlling or eradicatinginsect infestation are desirable in many instances, in particular whencrops of commercial interest are at issue. The most widely usedenvironmentally-sensitive insect inhibitory formulations developed inrecent years have been composed of microbial pest control agents derivedfrom the bacterium Bacillus thuringiensis. B. thuringiensis is wellknown in the art, and is characterized morphologically as aGram-positive bacterium that produces crystal proteins or inclusionbodies which are aggregations of proteins specifically active againstcertain orders and species of insects. Many different strains of B.thuringiensis have been shown to produce insect inhibitory crystalproteins. Compositions including B. thuringiensis strains which produceinsect inhibitory proteins have been commercially available and used asenvironmentally-acceptable pest control agents because they are quitetoxic to the specific target insect, but are harmless to plants andother non-targeted organisms.

[0006] There are several B.t. crystal protein categories establishedbased on primary structure information and the degree of proteinsimilarities to one another. Over the past decade, research on thestructure and function of B. thuringiensis crystal proteins has coveredall of the major categories, and while these proteins differ in specificstructure and function, general similarities in the structure andfunction are assumed. Based on the accumulated knowledge of B.thuringiensis insect inhibitory proteins, a generalized mode of actionfor B. thuringiensis insect inhibitory proteins has been created andincludes: ingestion by the insect, solubilization in the insect midgut(a combination of stomach and small intestine), resistance to digestiveenzymes sometimes with partial digestion actually “activating” theinsect inhibitory protein, binding to the midgut cells, formation of apore in the insect cells and the disruption of cellular homeostasis(English and Slatin, 1992).

[0007] Many of the δ-endotoxins are related to various degrees bysimilarities in their amino acid sequences. Historically, the proteinsand the genes which encode them were classified based largely upon theirspectrum of insect inhibitory activity. The review by Schnepf et al.(Microbiol. Mol. Biol. Rev. (1998) 62:775-806) discusses the genes andproteins that were identified in B. thuringiensis prior to 1998, andsets forth the most recent nomenclature and classification scheme asapplied to B. thuringiensis insect inhibitory genes and proteins. Usingolder nomenclature classification schemes, cry1 genes were deemed toencode lepidopteran-inhibitory Cry1 proteins, cry2 genes were deemed toencode lepidopteran- and dipteran-inhibitory Cry2 proteins, cry3 geneswere deemed to encode coleopteran-inhibitory Cry3 proteins, and cry4genes were deemed to encode dipteran-inhibitory Cry4 proteins. However,new nomenclature systematically classifies the Cry proteins based uponamino acid sequence homology rather than upon insect targetspecificities. The classification scheme for many known proteins, notincluding allelic variations in individual proteins, includingdendograms and full Bacillus thuringiensis protein lists is summarizedand regularly updated athttp://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html.

[0008] Most of the nearly 200 B.t. crystal proteins presently known havesome degree of lepidopteran activity associated with them. The largemajority of Bacillus thuringiensis insect inhibitory proteins which havebeen identified do not have coleopteran controlling activity. Therefore,it is particularly important, at least for commercial purposes, toidentify additional coleopteran specific insect inhibitory proteins.

[0009] The B.t. proteins which have been identified as havingcoleopteran-inhibitory activity are either related to the Cry3 proteinclass, or are greater than about 74 kDa in size. (Berhnard, 1986;Donovan et al., 1988, 1992; Herrnstadt et al., 1986; Hofte et al., 1987,1989; Kreig et al., 1983, 1984, 1987; McPherson et al., 1988; Sekar etal., 1987; Sick et al., 1990; U.S. Pat. No. 4,766,203; U.S. Pat. No.4,771,131; U.S. Pat. No. 4,797,279; U.S. Pat. No. 4,910,016; U.S. Pat.No. 4,966,155; U.S. Pat. No. 4,966,765; U.S. Pat. No. 4,999,192; U.S.Pat. No. 5,006,336; U.S. Pat. No. 5,024,837; U.S. Pat. No. 5,055,293;U.S. Pat. No. 6,023,013; European Pat. Appl. Publ. No. 0318143; Eur.Pat. Appl. Publ. No. 0324254; Eur. Pat. Appl. Publ. No. 0382990; PCTIntl. Pat. Appl. Publ. No. WO 90/13651; Intl. Pat. Appl. Publ. No. WO91/07481).

[0010] U.S. Pat. No. 6,063,756 disclosed Bacillus thuringiensis strainscomprising novel crystal proteins which exhibit insect inhibitoryactivity against coleopteran insects including red flour beetle larvae(Tribolium castaneum) and Japanese beetle larvae (Popillia japonica).Also disclosed therein are novel B. thuringiensis genes, designatedcryET33 and cryET34, which encode the coleopteran-inhibitory crystalproteins ET33 and ET34. cryET33 encodes the CryET33 (29-kDa) crystalprotein, and the cryET34 gene encodes the 14-kDa CryET34 crystalprotein. Also disclosed therein are methods of making and usingtransgenic cells comprising the novel nucleic acid sequences of theinvention.

[0011] Rupar et al. (WO00/066,742; PCT/US00/12136) describe still otherexpression systems isolated from Bacillus thuringiensis strains whichexpress proteins, which, when present in approximately equimolarconcentrations, exhibit Coleopteran insecticidal activity. Inparticular, a binary toxin system referred to as CryET80 and CryET76,ET76 being about 44 kDa and ET80 being about 14 kDa, are effective incontrolling corn rootworms.

[0012] Narva et al. (U.S. patent application Ser. No. 09/378,088;WO01/14417(A2); PCT/US00/22942) disclose yet at least one othercoleopteran inhibitory binary toxin exhibiting corn rootworm controllingbioactivity, isolated from Bacillus thuringiensis, and describe theconstruction of a fusion between the two components of the toxin, butfailed do demonstrate any bioactivity of this fusion.

[0013] It would be useful to provide a protein to plants which exhibitscoleopteran-inhibitory activity, which is less than about 74-kDa insize, which is expressed from a single open reading frame in order to,at least in plants, ensure simultaneous expression, and in particular inplants, in consideration of conservation of the genetic elements, createan easier means for breeding purposes.

SUMMARY OF THE INVENTION

[0014] The present invention discloses novel coleopteran-inhibitoryproteins and fusions of these proteins which also surprisingly exhibitinsecticidal activity equivalent to the levels of activity exhibited bythe native proteins, as well as novel nucleic acid sequences whichencode these proteins. Some of the improvements in the art claimed anddisclosed herein include the expression of a nucleic acid sequenceencoding two-component toxins in planta driven by one promoter, whereinsaid sequence encodes a fusion of the two components which allows forconservation of genetic elements and ensures expression of the wholetoxin within one cell at the same time. Also disclosed are methods ofmaking and using said nucleic acid sequence in the development oftransgenic plant cells containing the nucleic acid sequences disclosedherein.

[0015] One aspect of the present invention includes the amino acid andnucleic acid sequences as set forth in SEQ ID:2 and SEQ ID:4,respectively corresponding to Bacillus thuringiensis insecticidalcrystal proteins tIC100 and tIC101. These proteins can be isolated andpurified after expression from such nucleic acids as those set forth inSEQ ID NO:1 and SEQ ID NO:3.

[0016] Another aspect of the present invention includes novel amino acidand nucleic acid sequences resulting from the fusion of the CryET33coding sequence in frame with the CryET34coding sequence (SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17), and novel amino acid andnucleic acid sequences resulting from the fusion of the CryET34 codingsequence in frame with the CryET33 coding sequence (SEQ ID NO:19, SEQ IDNO:21). The present invention also includes novel amino acid and nucleicacid sequences resulting from the fusion of the tIC100 coding sequencein frame with the tIC101 coding sequence (SEQ ID NO:7, SEQ ID NO:9), andamino acid and nucleic acid sequences resulting from the fusion of thetIC101 coding sequence in frame with the tIC100 coding sequence (SEQ IDNO:5). Given the similarity in size, sequence, and insect inhibitoryspectrum activity between the CryET33 and tIC100 proteins, as well asbetween the CryET34 and tIC101 proteins, fusions comprising the CryET33sequence in frame with the tIC101 sequence and the tIC100 sequence inframe with the CryET34 sequence are also envisioned. tIC100 and tIC101are each believed to be novel proteins which have been shown to exhibitColeopteran insecticidal activity when present together in a compositionin about equimolar ratios.

[0017] Another aspect of the present invention relates to a recombinantvector comprising a nucleic acid sequence encoding a CryET33/CryET34,CryET34/CryET33, tIC100/tIC101, tIC101/tIC100, CryET33/tIC101, ortIC100/CryET34 fusion protein, wherein the sequence encoding the proteinis within a single expression cassette and its expression is controlledor driven by a single promoter. A recombinant host cell transformed withsuch a recombinant vector, and a biologically pure culture of therecombinant host cell so transformed are also exemplified herein. Thehost cell can be a plant cell or a bacterium, the bacterium preferablybeing a B. thuringiensis bacterium. In addition, a recombinant vectorcomprising a nucleic acid sequence encoding the tIC100 and the tIC101proteins from within a single operon is also disclosed. A recombinanthost cell transformed with such a recombinant vector and a biologicallypure culture of the recombinant host cell so transformed are alsoexemplified herein. The host cell can be a plant cell or a bacterium,the bacterium preferably being a Pseudomonas or a B. thuringiensisspecies of bacterium.

[0018] The present invention discloses an isolated insecticidalpolypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, and SEQ ID NO:26. The isolated insecticidal polypeptide exhibitsinsecticidal activity when provided in an orally acceptable insect dietto a susceptible Coleopteran insect or Coleopteran insect larva. Theisolated insecticidal polypeptide exhibits insecticidal activity whenprovided in an orally administrable diet to a susceptible Coleopteraninsect or Coleopteran insect larva. The isolated insecticidalpolypeptide exhibits a preferred insect inhibitory activity against aColeopteran insect, and the preferred Coleopteran insect is a cottonboll weevil adult or a cotton boll weevil larva.

[0019] The insecticidal polypeptide can be formulated into a compositioncomprising an insecticidally effective amount of the polypeptide whereinthe composition is a bacterial cell which expresses the polypeptide froma polynucleotide sequence that encodes said polypeptide. The compositioncan be any of or a combination of a cell extract, a cell suspension, acell homogenate, a cell lysate, a cell supernatant, a cell filtrate, ora cell pellet. The bacterial cell composition is preferably a bacterialcell comprised of a bacterial species selected from the speciesconsisting of a Bacillus species, an Escherichia species, a Salmonellaspecies, an Agrobacterium species, and a Pseudomonas species ofbacterial cell. The more preferable bacterial cell composition can beselected from the group of bacterial cells containing a recombinantplasmid, the group of bacterial cells being selected from a sIC2000bacterial cell, a sIC2001 bacterial cell, a sIC2002 bacterial cell, asIC2003 bacterial cell, a sIC2006 bacterial cell, a sIC2007 bacterialcell, a sIC2008 bacterial cell, and a sIC2010 bacterial cell.

[0020] The insecticidal composition can be an insecticidally effectiveamount of any of the polypeptides disclosed herein and can be formulatedas a powder, dust, pellet, granule, spray, emulsion, colloid, orsolution. The composition can be prepared by desiccation,lyophilization, homogenization, extraction, filtration, centrifugation,sedimentation, or concentration. The composition should contain theinsecticidal polypeptide present in a concentration of from about 0.001%to about 99% by weight.

[0021] The present invention also discloses an isolated polynucleotidesequence encoding an insecticidal polypeptide, wherein saidpolynucleotide is selected from the group consisting of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, and SEQ ID NO:25, and biologically functional equivalentsthereof. These polynucleotide sequences encode polypeptides whichexhibit Coleopteran insecticidal activity when provided orally to asusceptible Coleopteran insect or Coleopteran insect larva. Thesepolynucleotide sequences encode polypeptides which exhibit Coleopteraninsecticidal activity when provided in an orally administrable diet orcomposition to a Coleopteran insect or Coleopteran insect larva. Thesepolynucleotide sequences or variants of these sequences which encode thepolypeptides as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26 orfunctional equivalents of these polypeptides are useful for controllingColeopteran insects, in particular cotton boll weevils and cotton bollweevil larvae. A further useful polynucleotide sequence which isdisclosed herein is a polynucleotide sequence which is or iscomplementary to one or more of the polynucleotide sequences as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25 which hybridizesunder stringent conditions as defined herein to a polynucleotidesequence which is complementary to or which encodes a polypeptideselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQID NO:26, and biologically functional equivalents thereof.

[0022] Nucleic Acid and Amino Acid Sequences

[0023] The present invention concerns nucleic acid sequences that can beisolated from Bacillus thuringiensis strains, or synthesized entirely invitro using methods that are well-known to those of skill in the art. Asused herein, the term “nucleic acid sequence” refers to a DNA moleculethat has been isolated free of total genomic DNA of a particularspecies. Therefore, a nucleic acid sequence encoding a crystal proteinor a fusion of crystal proteins refers to a DNA molecule that containscrystal protein coding sequences yet is isolated away from, or purifiedfree from, total genomic DNA of the species from which the nucleic acidsequence is obtained, which in the instant case is the genome of theGram-positive bacterial genus, Bacillus, and in particular, the speciesof Bacillus known as B. thuringiensis. Also included within the term“nucleic acid sequence”, are recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

[0024] Similarly, a nucleic acid sequence comprising an isolated orpurified crystal protein-encoding gene or a nucleic acid sequenceencoding a fusion of crystal proteins refers to a nucleic acid sequencewhich may include, in addition to peptide encoding sequences, certainother elements such as, regulatory sequences, isolated substantiallyaway from other naturally occurring genes or protein-encoding sequences.In this respect, the term “gene” is used for simplicity to refer to afunctional protein-, polypeptide- or peptide-encoding unit. As will beunderstood by those in the art, this functional term includes bothgenomic sequences, operon sequences and smaller engineered genesequences that express, or may be adapted to express, proteins,polypeptides or peptides.

[0025] “Isolated substantially away from other coding sequences” meansthat the gene of interest, in this case, a gene encoding a bacterialcrystal protein or bacterial crystal protein fusion, forms thesignificant part of the coding region of the nucleic acid sequence, andthat the nucleic acid sequence does not contain large portions ofnaturally-occurring coding sequences, such as large chromosomalfragments or other functional genes or operon coding regions. Of course,this refers to the nucleic acid sequence as originally isolated, anddoes not exclude genes, recombinant genes, synthetic linkers, or codingregions later added to the sequence by the hand of man.

[0026] In particular embodiments, the invention comprises isolatednucleic acid sequences selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, and SEQ ID NO:31. The invention also is directed torecombinant vectors incorporating nucleic acid sequences that encode aprotein or fusion protein that includes within its amino acid sequencean amino acid sequence comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.

[0027] The term “a sequence essentially as set forth in SEQ ID NO:2”,for example, means that the sequence substantially corresponds to aportion of the sequence of SEQ ID NO:2 and has relatively few aminoacids that are not identical to, or are not biologically functionalequivalents of, the amino acids of any of the sequences contemplatedherein. The term “biologically functional equivalent” is well understoodin the art and is further defined in detail herein. Accordingly, aminoacid sequences that have between about 70% and about 80%, or morepreferably between about 81% and about 90%, or even more preferablybetween about 91% and about 99% amino acid sequence identity to eachother likely are functional equivalents of each other if each amino acidsequence exhibits some measurable activity such as insecticidal activityand each amino acid sequence provides comparable measurable activitywhen present in equimolar or substantially identical equimolar amounts.Functional equivalence to the amino acid sequences of SEQ ID NO:2 whencombined in equimolar ratios with SEQ ID NO:4, for example, will beamino acid sequences which are from about 70% to about 80% identical to,or more preferably from about 81% to about 90% identical to, or evenmore preferably from about 91% to about 99% identical to SEQ ID NO:2 andSEQ ID NO:4 and also exhibit substantially the same level ofinsecticidal activity on a weight to weight basis or a mole to molebasis.

[0028] Nucleic acid sequences can also be functionally equivalent toeach other. In this case, a first nucleic acid sequence encoding a firstpeptide can be functionally equivalent to a second nucleic acid sequenceencoding the same first peptide, primarily because of the redundancy ofthe genetic code. The second nucleic acid sequence can also befunctionally equivalent to the first nucleic acid sequence if thepeptide encoded by the second nucleic acid sequence is substantiallysimilar to the first peptide, for example exhibiting from about 70% toabout 80% identity to, or more preferably from about 81% to about 90%identity to, or even more preferably from about 91% to about 99%identity to the first peptide encoded by the first nucleic acidsequence, in particular, if the first and the second peptides exhibitsubstantially the same level of measurable activity on a weight toweight basis or on a mole to mole basis.

[0029] The nucleic acid sequences of the present invention encompasssequences encoding biologically-functional, equivalent peptides. Suchsequences may arise as a consequence of codon degeneracy and functionalequivalency that are known to occur naturally within nucleic acidsequences and the proteins thus encoded. Alternatively,functionally-equivalent proteins or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by manmay be introduced through the application of site-directed mutagenesistechniques, e.g., to introduce improvements to the antigenicity of theprotein or to test mutants in order to examine activity at the molecularlevel.

[0030] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding regionor may include various internal sequences, i.e., introns, which areknown to occur within genes.

[0031] The nucleic acid sequences of the present invention, regardlessof the length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding sequences, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid sequenceof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, nucleic acid fragments may beprepared that include a short contiguous stretch encoding either of thepeptide sequences disclosed in SEQ ID NO:2 or SEQ ID NO:4, or that areidentical to or complementary to nucleic acid sequences which encode anyof the peptides disclosed in SEQ ID NO:2 or SEQ ID NO:4, andparticularly those nucleic acid sequences disclosed in SEQ ID NO: 1 orSEQ ID NO:3. For example, nucleic acid sequences consisting of fromabout 14 nucleotides, and up to about 10,000, or to about 5,000, or toabout 3,000, or to about 2,000, or to about 1,000, or to about 500, orto about 200, or to about 100, or to about 50, and to about 14 basepairs in length (including all intermediate lengths) are alsocontemplated to be useful.

[0032] It will be readily understood that “intermediate lengths”, inthese contexts, means any length between the quoted ranges, such as 18,19, 20, 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100,101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integersthrough the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000;and up to and including sequences of about 5200 nucleotides and thelike.

[0033] It will also be understood that this invention is not limited tothe particular nucleic acid sequences which encode peptides of thepresent invention, or which encode the amino acid sequences of, forexample, SEQ ID NO:2 or SEQ ID NO:4, including those nucleic acidsequences which are particularly disclosed in SEQ ID NO:1 or SEQ IDNO:3. Recombinant vectors and isolated nucleic acid sequences may,therefore, variously include the peptide-coding regions themselves,coding regions bearing selected alterations or modifications in thebasic coding region, or they may encode larger polypeptides thatnevertheless include these peptide-coding regions or may encodebiologically functional equivalent proteins or peptides that havevariant amino acids sequences.

[0034] If desired, one may also prepare fusion proteins and peptidesother than those disclosed and claimed herein, e.g., where thepeptide-coding regions are aligned within the same expression unit withother proteins or peptides having desired functions, such as forpurification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

[0035] Recombinant vectors form further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the nucleic acid sequence,whether encoding a full length protein or smaller peptide, is positionedunder the control of a promoter. The promoter may be in the form of thepromoter that is naturally associated with a gene encoding peptides ofthe present invention, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding sequence, for example, usingrecombinant cloning and/or thermal amplification technology, inconnection with the compositions disclosed herein.

[0036] Nucleic Acid Sequences as Hybridization Probes and Primers

[0037] In addition to their use in directing the expression of crystalfusion proteins or peptides of the present invention, the nucleic acidsequences contemplated herein also have a variety of other uses. Forexample, they also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsequences that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous nucleic acid sequenceof, for example, SEQ ID NO:1 or SEQ ID NO:3 will find particularutility. Longer contiguous identical or complementary sequences, e.g.,those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000 basepairs, etc. (including all intermediate lengths and up to and includingthe full-length sequence of 5200 base pairs) will also be of use incertain embodiments.

[0038] The ability of such nucleic acid probes to specifically hybridizeto crystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

[0039] Nucleic acid molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so, identical or complementary to nucleic acidsequences of, for example, SEQ ID NO:1 or SEQ ID NO:3, are particularlycontemplated as hybridization probes for use in, e.g., Southern andNorthern blotting. Smaller fragments will generally find use inhybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 10-14 andabout 100 or 200 nucleotides, but larger contiguous complementaritystretches may be used, according to the length complementary sequencesone skilled in the art wishes to detect.

[0040] Of course, fragments of nucleic acids may also be obtained byother techniques such as, e.g., by mechanical shearing or by restrictionenzyme digestion. Small nucleic acid sequences or fragments may bereadily prepared by, for example, directly synthesizing the fragment bychemical means, as is commonly practiced using an automatedoligonucleotide synthesizer. Also, fragments may be obtained byapplication of nucleic acid reproduction technology, such as the thermalamplification technology of U.S. Pat. Nos. 4,683,195 and 4,683,202, byintroducing selected sequences into recombinant vectors for recombinantproduction, and by other recombinant DNA techniques generally known tothose of skill in the art of molecular biology.

[0041] Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. Such selectiveconditions tolerate little, if any, mismatch between the probe and thetemplate or target strand, and would be particularly suitable forisolating crystal protein-encoding DNA sequences. Detection of DNAsequences via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses. Teachings such asthose found in the texts of Maloy et al., 1993; Segal 1976; Prokop,1991; and Kuby, 1991, are particularly relevant.

[0042] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0043] In certain embodiments, it will be advantageous to employ nucleicacid sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavid/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel such as fluorescein or related molecules, or an enzyme tag such asurease, jellyfish green fluorescent protein or variants thereof,alkaline phosphatase, or peroxidase, instead of radioactive or otherenvironmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

[0044] In general, it is envisioned that the hybridization probesdescribed herein will be useful both as reagents in solutionhybridization as well as in embodiments employing a solid phase. Inembodiments involving a solid phase, the test DNA (or RNA) is adsorbedor otherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the incorporated label.

[0045] Recombinant Vectors and Crystal Protein Expression

[0046] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA sequence under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a DNA sequence encoding a crystalprotein or peptide in its natural environment. Such promoters mayinclude promoters normally associated with other genes, and/or promotersisolated from any bacterial, viral, eukaryotic, or plant cell.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA sequence in the cell type, organism,or even animal, chosen for expression. Those of skill in the art ofmolecular biology generally know the use of promoter and cell typecombinations for protein expression, for example, see Sambrook et al.,1989. The promoters employed may be constitutive, or inducible, and canbe used under the appropriate conditions to direct high level expressionof the introduced DNA sequence, such as is advantageous in thelarge-scale production of recombinant proteins or peptides. Appropriatepromoter systems contemplated for use in high-level expression include,but are not limited to, the Pichia expression vector system (PharmaciaLKB Biotechnology).

[0047] In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA sequences willmost often be used, with DNA sequences encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA sequences to direct the expression of crystalpeptides or epitopic core regions, such as may be used to generateanti-crystal protein antibodies, also falls within the scope of theinvention. DNA sequences that encode peptide antigens from about 8 toabout 50 amino acids in length, or more preferably, from about 8 toabout 30 amino acids in length, or even more preferably, from about 8 toabout 20 amino acids in length are contemplated to be particularlyuseful. Such peptide epitopes may be amino acid sequences which comprisecontiguous amino acid sequences from, for example, SEQ ID NO:2 or SEQ IDNO:4.

[0048] Crystal Protein Transgenes and Transgenic Plants

[0049] In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid sequenceencoding one of the novel crystal proteins of the present invention. Theprocess of producing transgenic plants is well-known in the art. Forexample, the method comprises, in general, transforming a suitable hostcell with a DNA sequence which contains a promoter operatively linked toa coding region that encodes, for example, a B. thuringiensisCryET33/CryET34 crystal fusion protein, or for example, a B.thuringiensis CrytIC100 or CrytIC101 crystal protein, or combinations ofthereof. Such a coding region is generally operatively linked to atranscription-terminating region, whereby the promoter is capable ofdriving the transcription of the coding region in the cell, and henceproviding the cell the ability to produce the recombinant protein invivo. Alternatively, in instances where there is a desire to control,regulate, or decrease the amount of a particular recombinant crystalprotein expressed in a particular transgenic cell, the invention alsoprovides for the expression of crystal protein antisense mRNA. The useof antisense mRNA as a means of controlling or decreasing the amount ofa given protein of interest in a cell is well-known in the art.

[0050] Further embodiments disclosed herein include expression of theproteins tIC100 and tIC101 (SEQ ID NO:2 and SEQ ID NO:4, respectively)in a plant, alone or in combination. For example, tIC100 cold beexpressed in one plant from an expression cassette which is linkedphysically to a second cassette expressing tIC101 so that both proteinsare expressed in the same plant. Each protein could be expressed in aplant from separate promoters but the coding sequences of each proteinbeing physically linked, for example, on the same chromosome.Alternatively, each protein could be expressed in a plant from separatepromoters but the coding sequences of each protein are not physicallylinked, for example, but the expression cassettes containing thepromoter operably linked to the coding sequence are instead present inthe same plant cell but on different chromosomes, so that Mendeliansegregation can be achieved if desired. Alternatively, these proteinscould be expressed from gene sequences transformed into the chloroplastgenome, or from autonomously replicating epigenetic elements presentwithin the chloroplast stroma. Yet another alternative embodimentcomprises expression of these proteins as a fusion protein, the carboxyterminus of one of these proteins being linked either directly, by aflexible amino acid sequence linker, or by an amino acid sequence linkercomprising a sequence susceptible to protease or autocatalytic cleavageupon expression or subcellular localization of the expression productfusion protein, or allowing cleavage of the linker region upon ingestionand localization of the fusion protein to the midgut of a target insectlarvae, resulting in the release of the two proteins into the cellularmilieu or into the midgut digestive fluids in approximately equimolarproportions and allowing the two proteins to be activated as abiologically active insecticidal crystal protein. Still as anotheralternative embodiment, tIC100 and tIC101 can be mixed with otherrelated binary toxins in various compositions or proportions in order toachieve a broader host range, improved insecticidal specificity, orimproved insecticidal activity. For example, tIC101 could be presentedto a coleopteran insect in approximately equimolar concentrations withET33, resulting in a surprisingly effective coleopteran insecticidaltoxin. tIC100 could be presented to a coleopteran insect inapproximately equimolar concentrations with ET34 also resulting in asurprisingly effective coleopteran insecticidal toxin. Alternatively,these toxin components could be presented to a susceptible coleopteraninsect in the form of fusions resulting in a surprisingly effectivecoleopteran insecticidal toxin. In yet another embodiment, these toxinscould be presented together (tIC100, tIC101, ET33, and ET34, together orin various compositions exhibiting insecticidal activity) to acoleopteran insect in a composition which facilitates insect resistancemanagement practices. Alternatively, these toxin compositions could beprovided with other coleopteran toxins such as for example Cry22, Cry3,or ET70 to provide surprisingly effective compositions for increasinginsect resistance management. Additional resistance management practicescontemplated herein include compositions of insecticidal proteinsdisclosed herein along with non-Bacillus thuringiensis insecticidalproteins, for example, insecticidal proteins isolatable from otherspecies known in the art which have been shown to be insecticidal suchas Xenorhabdus and Photorhabdus species of bacteria.

[0051] Another aspect of the invention comprises transgenic plants thatexpress one or more genes or gene sequences encoding one or more of thenovel polypeptide compositions disclosed herein. As used herein, theterm “transgenic plant” is intended to refer to a plant that hasincorporated DNA sequences, including but not limited to genes which areperhaps not normally present, DNA sequences not normally transcribedinto RNA or translated into a protein (“expressed”), or any other genesor DNA sequences which one desires to introduce into the non-transformedplant, such as genes which may normally be present in thenon-transformed plant but which one desires to either geneticallyengineer or to have altered expression.

[0052] Means for transforming a plant cell and the preparation of atransgenic cell line are well-known in the art, and are discussedherein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes)and DNA sequences for use in transforming such cells will, of course,generally comprise either the operons, genes, or gene-derived sequencesof the present invention, either native, or synthetically-derived, andparticularly those encoding the disclosed crystal proteins. These DNAconstructs can further include structures such as promoters, enhancers,introns, terminators, operators, polyadenylation signals, or other genesequences which have positively- or negatively-regulating activity uponthe particular genes of interest as desired. The DNA sequence or genemay encode either a native or modified crystal protein, which will beexpressed in the resultant recombinant cells, and/or which will impartan improved phenotype to the regenerated plant.

[0053] Such transgenic plants may be desirable for increasing the insectinhibitory resistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a nucleic acid sequence comprising oneor more of the sequences discussed herein and encoding crystal proteinwhich is toxic to Coleopteran insects. Particularly preferred plantsinclude corn, cotton, potato, soybean, canola, tomato, turf grasses,wheat, vegetables, ornamental plants, fruit trees, and the like.

[0054] In a related aspect, the present invention also encompasses aseed produced by the transformed plant, a progeny from such seed, and aseed produced by the progeny of the original transgenic plant, producedin accordance with the above process. Such progeny and seeds will have acrystal protein-encoding nucleic acid sequence stably incorporated intotheir genome, and such progeny plants will preferably inherit the traitsconferred by the nucleic acid sequence in Mendelian fashion. All suchtransgenic plants having incorporated into their nuclear genome nucleicacid sequences comprising one or more of the sequences discussed hereinand encoding one or more crystal proteins or polypeptides are aspects ofthis invention.

[0055] Plants comprising cells comprising chloroplasts transformed tocontain nucleic acid sequences encoding the proteins of the presentinvention are also contemplated. Such plants would not be expected topass these traits to their progeny plants or seeds through Mendelianfashion, but instead would pass on these traits to progeny throughmaternal transmission means well known in the art.

[0056] Site-Specific Mutagenesis

[0057] Site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying nucleic acid sequence. The technique further provides a readyability to prepare and test sequence variants, for example,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the originalnucleic acid sequence. Means for site-specific mutagenesis provides forthe production of nucleic acid sequence variants through the use ofspecific synthetic oligonucleotide sequences which hybridize to thetarget nucleic acid sequence intended to be altered. Such syntheticoligonucleotides comprise the nucleic acid sequence of the desiredmutation or sequence variant at the target site sequence, as well as asufficient number of nucleotides complementary to the sequences flankingthe target site sequence, said synthetic oligonucleotide acting as aprimer sequence of sufficient size and sequence complexity to form astable heteroduplex with the target nucleic acid sequence at theintended target site and generally flanking both sides of the intendedtarget site sequence. Typically, a primer of about 17 to 25 nucleotidesin length is preferred, with about 5 to 10 residues on both sides of thesequence being altered. The target site intended to be altered to formthe variant sequence could incorporate either a single nucleotide, oralternatively could be two nucleotides or even more than two nucleotideseach adjacent to each other or interspersed throughout the syntheticmutagenesis oligonucleotide sequence. One skilled in the art wouldreadily recognize that a single nucleotide sequence change would requirea synthetic oligonucleotide which would be considerably shorter inlength than would a synthetic oligonucleotide sequence which is intendedfor use in incorporating two or more changes to the original nucleotidesequence, and therefore would generally, although not always, requirelonger sequences of complementarity to the sequences flanking theintended target site sequence(s).

[0058] Crystal Protein Screening and Detection Kits

[0059] The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides. Akit may contain one or more antibodies of the present invention, and mayalso contain reagent(s) for detecting an interaction between a sampleand an antibody of the present invention. The provided reagent(s) can beradio-, fluorescently- or enzymatically-labeled. The kit can contain aknown radio-, flourescent-, hapten-, or enzyme-labeled agent capable ofbinding or interacting with a nucleic acid, protein or antibody of thepresent invention.

[0060] The reagent(s) of the kit can be provided as a liquid solution,attached to a solid support or as a dried powder. Preferably, when thereagent(s) are provided in a liquid solution, the liquid solution is anaqueous solution. Preferably, when the reagent(s) provided are attachedto a solid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent(s) provided are a dry powder, the powder can be reconstituted bythe addition of a suitable solvent, that may be provided.

[0061] In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

[0062] In general, the detection of immunocomplex formation is quitewell known in the art and may be achieved through the application ofnumerous approaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

[0063] For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

[0064] The container will generally include a vial into which theantibody, antigen or detection reagent may be placed, and preferablysuitably subsequently distributed into samples intended for analysis.The kits of the present invention will also typically include a meansfor containing the antibody, antigen, and reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained.

[0065] Biological Functional Equivalents

[0066] Modification and changes may be made in the structure of thepeptides of the present invention and nucleic acid sequences whichencode them and still obtain a functional molecule that encodes aprotein or peptide with desirable characteristics. The following is adiscussion based upon changing the amino acids of a protein to create anequivalent, or even an improved, second-generation molecule. Inparticular embodiments of the invention, mutated or variant crystalproteins are contemplated to be useful for increasing the insectinhibitory activity of the protein, and consequently preferablyincreasing the insect inhibitory activity and/or expression of therecombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 1. TABLE 1 Amino Acids and Corresponding CodonsAmino Acids * ** Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGCUGU Aspartate Asp D GAC GAU Glutamate Glu E GAA GAG Phenylalanine Phe FUUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU IsoleucineIle I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UAA UUG CUA CUC CUGCUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCCCCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0067] For example, certain amino acids, known as conservative aminoacids, may be substituted for other amino acids in a protein structurewithout appreciable loss of interactive binding capacity with structuressuch as, for example, antigen-binding regions of antibodies or bindingsites on substrate molecules. Since it is the interactive capacity andnature of a protein that defines the protein's biological functionalactivity, certain amino acid sequence substitutions can be made in aprotein sequence, and, of course, its underlying DNA coding sequence,and nevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in thepeptide sequences of the disclosed compositions, or corresponding DNAsequences which encode said peptides without appreciable loss of theirbiological utility or activity.

[0068] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0069] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte andDoolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0070] It is known in the art that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within +/−0.2 are preferred, those which are within +/−0.1are particularly preferred, and those within +/−0.05 are even moreparticularly preferred.

[0071] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, discloses that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

[0072] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1); glutamate(+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); tryptophan (−3.4).

[0073] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within +/−0.2 are preferred, those whichare within +/−0.1 are particularly preferred, and those within +/−0.05are even more particularly preferred.

[0074] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

[0075] Crystal Protein Insect Inhibitory Compositions and Methods of Use

[0076] The inventors contemplate that the crystal protein compositionsdisclosed herein will find particular utility as insect inhibitory orinsecticidal compositions for topical and/or systemic application tofield crops, grasses, fruits and vegetables, and ornamental plants. In apreferred embodiment, the biological insect inhibitory or insecticidalcomposition comprises an oil flowable suspension of bacterial cellswhich expresses a novel crystal protein disclosed herein. Any bacterialhost cell expressing the novel nucleic acid sequences disclosed hereinand producing a crystal protein is contemplated to be useful, such as B.thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.

[0077] In another embodiment, the biological insect inhibitorycomposition comprises a water dispersible granule. This granulecomprises bacterial cells which express one or more of the novel crystalproteins disclosed herein. Bacteria such as B. thuringiensis, B.megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformedwith a DNA sequence disclosed herein and expressing one or more of thecrystal proteins are also contemplated to be useful.

[0078] In a third embodiment, the biological insect inhibitory orinsecticidal composition comprises a wettable powder, dust, pellet, orcolloidal concentrate. This powder comprises bacterial cells whichexpress one or more of the novel crystal proteins disclosed herein.Bacteria such as B. thuringiensis, B. megaterium, B. subtilis, E. coli,or Pseudomonas spp. cells transformed with one or more of the nucleicacid sequences disclosed herein and expressing the crystal protein arealso contemplated to be useful. Such dry forms of the insect inhibitorycompositions may be formulated to dissolve immediately upon wetting, oralternatively, dissolve in a controlled-release, sustained-release, orother time-dependent manner.

[0079] In a fourth embodiment, the biological insect inhibitory orinsecticidal composition comprises an aqueous suspension of bacterialcells such as those described above which express the crystal protein.Such aqueous suspensions may be provided as a concentrated stocksolution which is diluted prior to application, or alternatively, as adiluted solution ready-to-apply. For methods involving application ofbacterial cells, the cellular host containing the crystal proteingene(s) may be grown in any convenient nutrient medium, where the DNAconstruct provides a selective advantage, providing for a selectivemedium so that substantially all or all of the cells retain the B.thuringiensis gene. These cells may then be harvested in accordance withconventional means. Alternatively, the cells can be treated prior toharvesting.

[0080] When the insect inhibitory or insecticidal compositions compriseintact B. thuringiensis cells expressing the protein of interest, suchbacteria may be formulated in a variety of ways. They may be employed aswettable powders, granules or dusts, by mixing with various inertmaterials, such as inorganic minerals (phyllosilicates, carbonates,sulfates, phosphates, and the like) or botanical materials (powderedcorncobs, rice hulls, walnut shells, and the like). The formulations mayinclude spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

[0081] Alternatively, the novel proteins discussed and claimed hereinmay be prepared by native or recombinant bacterial expression systems invitro and isolated for subsequent field application. Such protein may beeither in crude cell lysates, suspensions, colloids, etc., oralternatively may be purified, refined, buffered, and/or furtherprocessed, before formulating in an active biocidal formulation.Likewise, under certain circumstances, it may be desirable to isolatecrystals and/or spores from bacterial cultures expressing the crystalprotein and apply solutions, suspensions, or collodial preparations ofsuch crystals and/or spores as the active bioinsect inhibitorycomposition.

[0082] Regardless of the method of application, the amount of the activecomponent(s) is applied at an insect inhibitory- orinsecticidally-effective amount, which will vary depending on suchfactors as, for example, the specific coleopteran-inhibitory insects tobe controlled, the specific plant or crop to be treated, theenvironmental conditions, and the method, rate, and quantity ofapplication of the insect inhibitory-active composition.

[0083] The insect inhibitory compositions described may be made byformulating either the bacterial cell, crystal and/or spore suspension,or isolated protein component with the desired agriculturally-acceptablecarrier. The compositions may be formulated prior to administration inan appropriate means such as lyophilized, freeze-dried, dessicated, orin an aqueous carrier, medium or suitable diluent, such as saline orother buffer. The formulated compositions may be in the form of a dustor granular material, or a suspension in oil (vegetable or mineral), orwater or oil/water emulsions, or as a wettable powder, or in combinationwith any other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, e.g., inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in insecticide formulationtechnology; these are well known to those skilled in insecticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the insect inhibitory or insecticidalcomposition with suitable adjuvants using conventional formulationtechniques.

[0084] The insect inhibitory or insecticidal compositions of thisinvention are applied to the environment of the target coleopteraninsect, typically onto the foliage of the plant or crop to be protected,by conventional methods, preferably by spraying. The strength andduration of insect inhibitory or insecticidal application will be setwith regard to conditions specific to the particular pest(s), crop(s) tobe treated and particular environmental conditions. The proportionalratio of active ingredient to carrier will naturally depend on thechemical nature, solubility, and stability of the insect inhibitory orinsecticidal composition, as well as the particular formulationcontemplated.

[0085] Other application techniques, e.g., dusting, sprinkling, soaking,soil injection, seed coating, seedling coating, spraying, aerating,misting, atomizing, and the like, are also feasible and may be requiredunder certain circumstances such as e.g., insects that cause root orstalk infestation, or for application to delicate vegetation orornamental plants. These application procedures are also well-known tothose of skill in the art.

[0086] The insect inhibitory or insecticidal composition of theinvention may be employed in the method of the invention singly or incombination with other compounds, including and not limited to otherpesticides. The method of the invention may also be used in conjunctionwith other treatments such as surfactants, detergents, polymers ortime-release formulations. The insect inhibitory or insecticidalcompositions of the present invention may be formulated for eithersystemic or topical use.

[0087] The concentration of insect inhibitory or insecticidalcomposition which is used for environmental, systemic, or foliarapplication will vary widely depending upon the nature of the particularformulation, means of application, environmental conditions, and degreeof biocidal activity. Typically, the bioinsect inhibitory orinsecticidal composition will be present in the applied formulation at aconcentration of at least about 1% by weight and may be up to andincluding about 99% by weight. Dry formulations of the compositions maybe from about 1% to about 99% or more by weight of the composition,while liquid formulations may generally comprise from about 1% to about99% or more of the active ingredient by weight. Formulations whichcomprise intact bacterial cells will generally contain from about 10⁴ toabout 10⁷ cells/mg.

[0088] The insect inhibitory or insecticidal formulation may beadministered to a particular plant or target area in one or moreapplications as needed, with a typical field application rate perhectare ranging on the order of from about 50 g to about 500 g of activeingredient, or of from about 500 g to about 1000 g, or of from about1000 g to about 5000 g or more of active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0090]FIG. 1 is a schematic representation of a CryET33/CryET34 fusionprotein or a tIC100/tIC101 fusion protein linked together in frame by acoding sequence flanked by BamHI and NheI restriction sites and encodinga peptide sequence comprising Gly-Ser-Gly-Gly-Ala-Ser.

[0091]FIG. 2 is a schematic representation of a CryET34/CryET33 or atIC101/tIC100 fusion protein linked together in frame by a codingsequence flanked by BamHI and NheI restriction sites and encoding apeptide sequence comprising Gly-Ser-Gly-Gly-Ala-Ser.

[0092]FIG. 3 illustrates the results of a boll-weevil diet-overlaybioassay using a lepidopteran diet containing 0.1% stigmastanol forparticular CryET33/CryET34 (sIC200 and sIC2001) and tIC100/tIC101(sIC2006, sIC2007, and sIC2008) fusions.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0093] Some Advantages of the Invention

[0094] CryET33 and CryET34 incombination, and tIC100 and tIC101 incombination, are both two-component insecticidal protein systems, eachderived from different Bacillus thuringiensis strains, and requiringboth of the two proteins in an approximately equimolar ratio forbioactivity. Therefore, for either system to be effective, both proteinsneed to be present at the same time in order to confer protection toplant against coleopteran species insect infestation, and to boll weevilin particular. Each of the proteins are expressed from different codingsequences in their respective strain of Bt, however, each set ofproteins, i.e., CryET33 and CryET34 or CrytIC100 and CrytIC101, areexpressed together in Bt from a polycistronic messenger RNA transcribedfrom a single DNA sequence in which both coding sequences are linkedtogether in the genome. Therefore, the ability to express both proteinsas a single construct in plants would eliminate several problemsassociated with attempting to express two separate proteins concurrentlyin a transgenic plant system. The major advantage of the fusionconstruct is that both proteins will be expressed simultaneously as theyare under the control of a single promoter element. It is readilyapparent to one skilled in the art that the simultaneous expression oftwo constructs in planta to achieve equimolar ratios of the proteinswould be much more difficult than enabling the expression of oneconstruct. A corollary to this benefit then, is that expression of bothproteins in a single cassette would simplify subsequent breeding. In asubsequent breeding, the gene encoding both proteins would betransmitted to the progeny, or not at all, depending on whether theparent transmitting the gene was homozygous or heterozygous for thetrait at the locus of the gene within the chromosome containing thegene. However, by expressing the proteins from a common cassette, thesituation where only one gene of the pair is transmitted to subsequentgenerations will not occur if the genes are present on differentexpression cassettes and distal from each other on the same chromosomeor on different chromosomes, thus reducing the complexity of thebreeding of plants with the insect inhibitory protein expressed.Deletion of one gene of the pair by a crossover between elements incommon within an expression cassette would render this inhibitory orinsecticidal system of binary toxins derived from Bacillus thuringiensisineffective. A fusion protein would be protected from such anoccurrence, as both proteins would be expressed concurrently from withina single expression cassette. Expression as a fusion protein would alsoeliminate problems of gene silencing experienced with expression of twonovel proteins under the control of similar promoter elements.

[0095] Definitions

[0096] The following words and phrases have the meanings set forthbelow.

[0097] Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

[0098] Promoter: A recognition site on a DNA sequence or group of DNAsequences that provide an expression control element for a structuralgene and to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene.

[0099] Regeneration: The process of growing a plant from a plant cell(e.g., plant protoplast or explant).

[0100] Structural gene: A gene that is expressed to produce apolypeptide.

[0101] Susceptible insect larva: an insect larva which, upon havingorally ingested a sample of diet containing one or more of the proteinsof the present invention, the diet being either artificially produced orobtained from a plant tissue artificially coated with or expressing oneor more of the proteins of the present invention from a recombinant geneor genes, is growth inhibited as measured by failure to gain weight,molting cycle frequency inhibition, observed lethargic behaviour,reduction in frass production, or death in comparison to either 1) alarvae which does not exhibit any of these indications when feeding uponthe same diet provided to a susceptible larvae, or 2) a larvae which isfeeding upon a control diet which does not contain the one or moreproteins of the present invention.

[0102] Transformation: A process of introducing an exogenous DNAsequence (e.g., a vector, a recombinant DNA molecule) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

[0103] Transformed cell: A cell whose genetic composition, eitherchromosomal DNA or other naturally occurring intracellular DNA, has beenaltered by the introduction of an exogenous DNA molecule into thegenetic composition of that cell.

[0104] Transgenic cell: Any cell derived or regenerated from atransformed cell or derived from a transgenic cell. Exemplary transgeniccells include plant calli derived from a transformed plant cell andparticular cells such as leaf, root, stem, e.g., somatic cells, orreproductive (germ) cells obtained from a transgenic plant regeneratedfrom a transformed cell.

[0105] Transgenic plant: A plant or progeny thereof derived from atransformed plant cell or protoplast, wherein the plant DNA contains anintroduced exogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant containing an exogenous andartificially introduced DNA molecule within its own naturally occurringgenetic composition. However, it is thought more scientifically correctto refer to a regenerated plant or callus obtained from a transformedplant cell or protoplast as being a transgenic plant, and that usagewill be followed herein.

[0106] Vector: A DNA molecule capable of replication in a host celland/or to which another DNA sequence can be operatively linked so as tobring about replication of the attached sequence. A plasmid is anexemplary vector.

[0107] Probes and Primers

[0108] In another aspect, nucleic acid sequence information provided bythe invention allows for the preparation of relatively short DNA (orRNA) sequences having the ability to specifically hybridize to nucleicacid sequences of the selected polynucleotides disclosed herein. Inthese aspects, nucleic acid probes of an appropriate length are preparedbased on a consideration of the nucleic acid sequence encoding theselected crystal protein, e.g., a sequence such as that shown in SEQ IDNO:1 or SEQ ID NO:3. The ability of such nucleic acid probes tospecifically hybridize to a crystal protein-encoding nucleic acidsequence lends to those probes particular utility in a variety ofembodiments. Most importantly, the probes may be used in a variety ofassays for detecting the presence of complementary sequences in a givensample suspected of containing probe-complementary sequences.

[0109] In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying, modifying, ormutating a defined sequence of nucleic acid encoding a crystal proteinfrom B. thuringiensis using thermal amplification technology. Sequencesof related crystal protein genes from other species may also beamplified by thermal amplification technology using such primers.

[0110] In accordance with the present invention, a preferred nucleicacid sequence employed for hybridization studies or assays includessequences that are complementary to at least a 14 to 30 or so longnucleotide sequence derived from a crystal protein-encoding sequence,such as that shown in SEQ ID NO:1 or SEQ ID NO:3. A size of at least 14nucleotides in length helps to ensure that the fragment will be ofsufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 14 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtainedthrough probe hybridization. One will generally prefer to design nucleicacid molecules having sequence-complementary stretches of 14 to 20nucleotides, or even longer where desired. Such fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, by application of nucleic acid reproduction technology, such asthermal amplification technology disclosed in U.S. Pat. Nos. 4,683,195,and 4,683,202, herein incorporated by reference, or by excising selectedDNA fragments from recombinant plasmids containing appropriate insertsand suitable restriction sites.

[0111] Expression Vectors

[0112] The present invention contemplates expression vectors comprisinga polynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to an coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region.

[0113] As used herein, the term “operatively linked” means that apromoter is connected to an coding region in such a way that thetranscription of that coding region is controlled and regulated by thatpromoter. Means for operatively linking a promoter to a coding regionare well known in the art.

[0114] In a preferred embodiment, the recombinant expression of DNAsencoding the crystal proteins of the present invention is preferable ina Bacillus host cell. Preferred host cells include B. thuringiensis, B.megaterium, B. subtilis, and related bacilli, with B. thuringiensis hostcells being highly preferred. Promoters that function in bacteria arewell-known in the art. An exemplary and preferred promoter for theBacillus crystal proteins include any of the known crystal protein genepromoters, including the cryET33 and cryET34 gene promoters.Alternatively, mutagenized or recombinant crystal protein-encoding genepromoters may be engineered by the hand of man and used to promoteexpression of the novel gene sequences disclosed herein.

[0115] In an alternate embodiment, the recombinant expression of DNAsencoding the crystal proteins of the present invention is performedusing a transformed Gram-negative bacterium such as an E. coli orPseudomonas spp. host cell. Promoters which function in high-levelexpression of target polypeptides in E. coli and other Gram-negativehost cells are also well-known in the art.

[0116] Where an expression vector of the present invention is to be usedto transform a plant, a promoter is selected that has the ability todrive expression in plants. Promoters that function in plants are alsowell known in the art. Useful in expressing the polypeptide in plantsare promoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

[0117] A promoter is also selected for its ability to direct thetransformed plant cell's or transgenic plant's transcriptional activityto the coding region. Structural genes can be driven by a variety ofpromoters in plant tissues. Promoters can be near-constitutive, such asthe CaMV 35S promoter, or tissue-specific or developmentally specificpromoters affecting dicots or monocots.

[0118] Where the promoter is a near-constitutive promoter such as CaMV35S, increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

[0119] An exemplary tissue-specific promoter is the lectin promoter,which is specific for seed tissue. The Lectin protein in soybean seedsis encoded by a single gene (Le1) that is only expressed during seedmaturation and accounts for about 2 to about 5% of total seed mRNA. Thelectin gene and seed-specific promoter have been fully characterized andused to direct seed specific expression in transgenic tobacco plants(Vodkin et al., 1983; Lindstrom et al., 1990).

[0120] An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

[0121] A transgenic plant of the present invention produced from a plantcell transformed with a tissue specific promoter can be crossed with asecond transgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

[0122] Exemplary tissue-specific promoters are corn sucrose synthetase 1(Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989),corn light harvesting complex (Simpson, 1986), corn heat shock protein(Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen etal., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase(Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge etal., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), beanglycine rich protein 1 (Keller et al., 1989), CaMV 35s transcript (Odellet al., 1985) and Potato patatin (Wenzler et al., 1989). Preferredpromoters are the cauliflower mosaic virus (CaMV 35S) promoter and theS-E9 small subunit RuBP carboxylase promoter.

[0123] The choice of which expression vector and ultimately to whichpromoter a polypeptide coding region is operatively linked dependsdirectly on the functional properties desired, e.g., the location andtiming of protein expression, and the host cell to be transformed. Theseare well known limitations inherent in the art of constructingrecombinant DNA molecules. However, a vector useful in practicing thepresent invention is capable of directing the expression of thepolypeptide coding region to which it is operatively linked.

[0124] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described(Rogers et al., 1987). However, several other plant integrating vectorsystems are known to function in plants including pCaMVCN transfercontrol vector described (Fromm et al., 1985). Plasmid pCaMVCN(available from Pharmacia, Piscataway, N.J.) includes the caulifiowermosaic virus CaMV 35S promoter.

[0125] In preferred embodiments, the vector used to express thepolypeptide includes a selection marker that is effective in a plantcell, preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

[0126] RNA polymerase transcribes a coding DNA sequence through a sitewhere polyadenylation occurs. Typically, DNA sequences located a fewhundred base pairs downstream of the polyadenylation site serve toterminate transcription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

[0127] Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, the disclosures of which areincorporated herein by reference. Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

[0128] A variety of methods has been developed to operatively link DNAto vectors via complementary cohesive termini or blunt ends. Forinstance, complementary homopolymer tracts can be added to the DNAsequence to be inserted and to the vector DNA. The vector and DNAsequence are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

[0129] A coding region that encodes a polypeptide which confers insectinhibitory activity to a cell transformed to express the polypeptide ispreferably a sequence encoding a tIC100 and/or tIC101 polypeptide, or aCryET33/CryET34 fusion peptide, a CryET34/CryET33 fusion peptide, atIC100/tIC101 fusion peptide, a tIC101/tIC100 fusion peptide, aCryET33/tIC101 fusion peptide, or a tIC100/CryET34 fusion peptide, eachof these or combinations thereof being further defined as B.thuringiensis insecticidal crystal fusion proteins. For example, inpreferred embodiments, such a coding region has the nucleic acidsequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17 toencode a CryET33/CryET34 fusion, or a functional equivalent of thosesequences. Also, co-expression of coding sequences for either CryET33 ortIC100 along with either CryET34 or tIC101 are shown herein to conferinsect inhibitory activity to a plant or host cell.

[0130] Characteristics of the Novel Crystal Proteins

[0131] The present invention provides novel polypeptides that define awhole or a portion of tIC100, tIC101, CryET33/CryET34 fusions,tIC100/tIC101 fusions, CryET33/tIC101 fusions, and tIC100/CryET34fusions whereby the fusion proteins contain various linkers disclosedand claimed herein. Various calculated physical characteristics oftIC100, tIC101, CryET33/CryET34 fusions containing various linkers, andtIC100/tIC101 fusions containing various linkers are listed below. Thecalculated physical characteristics of tIC100/CryET34 and CryET33/tIC101fusions are not listed; however, such characteristics could be easilyderived using known methods by persons skilled in the art.

[0132] tIC100

[0133] tIC100 is a protein as set forth in SEQ ID NO:2 derived from acryptic B. thuringiensis DNA sequence. The cryptic tIC100 codingsequence as set forth in SEQ ID NO:1 is a part of an operon containingthe tIC101 coding sequence, and is adjacent to and upstream of thecoding sequence for tIC101. The cryptic sequence upstream of tIC101contains the complete coding sequence for tIC100 except that a singleguanosine residue at position 84 of the native cryptic tIC100 codingsequence as set forth in SEQ ID NO:27 causes the tIC100 coding sequenceto be out of frame. The frameshift was eliminated, as described inExample 6 herein, by removing the single guanosine residue at position84 to create the novel tIC100 coding sequence as set forth in SEQ IDNO:1, encoding the tIC100 protein as set forth in the translation in SEQID NO:1 and in the peptide sequence as set forth in SEQ ID NO:2, and asshown herein below. Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp1               5                   10 Tyr Met Lys Gly Met Tyr Gly AlaThr Ser Val Lys         15                  20 Ser Thr Tyr Asp Pro SerPhe Lys Val Phe Asn Glu 25                  30                  35 SerVal Thr Pro Gln Tyr Asp Val Ile Pro Thr Glu            40                  45 Pro Val Asn Asn His Ile Thr Thr LysVal Ile Asp     50                  55                  60 Asn Pro GlyThr Ser Glu Val Thr Ser Thr Val Thr                65                  70 Phe Thr Trp Thr Glu Thr Asp ThrVal Thr Ser Ala         75                  80 Val Thr Lys Gly Tyr LysVal Gly Gly Ser Val Ser 85                  90                  95 SerLys Ala Thr Phe Lys Phe Ala Phe Val Thr Ser            100                 105 Asp Val Thr Val Thr Val Ser Ala GluTyr Asn Tyr     110                 115                 120 Ser Thr ThrGlu Thr Thr Thr Lys Thr Asp Thr Arg                125                 130 Thr Trp Thr Asp Ser Thr Thr ValLys Ala Pro Pro         135                 140 Arg Thr Asn Val Glu ValAla Tyr Ile Ile Gln Thr 145                 150                 155 GlyAsn Tyr Asn Val Pro Val Asn Val Glu Ser Asp            160                 165 Met Thr Gly Thr Leu Phe Cys Arg GlyTyr Arg Asp     170                 175                 180 Gly Ala LeuIle Ala Ala Ala Tyr Val Ser Ile Thr                185                 190 Asp Leu Ala Asp Tyr Asn Pro AsnLeu Gly Leu Thr         195                 200 Asn Glu Gly Asn Gly ValAla His Phe Lys Gly Glu 205                 210                 215 GlyTyr Ile Glu Gly Ala Gln Gly Leu Arg Ser Tyr            220                 225 Ile Gln Val Thr Glu Tyr Pro Val AspAsp Asn Gly 230                 235                 240 Arg His Ser IlePro Lys Thr Tyr Ile Ile Lys Gly                 245                 250Ser Leu Ala Pro Asn Val Thr Leu Ile Asn Asp Arg        255                 260 Lys Glu Gly Arg 265

[0134] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 2.

[0135] Molecular weight=29239. Residues=268

[0136] Isoelectric point=4.79 TABLE 2 Amino Acid Composition of tIC100Residue Type Number of Residues Mole Percent In tIC100 Protein A = Ala15 5.597 B = Asx 0 0.000 C = Cys 1 0.373 D = Asp 16 5.970 E = Glu 145.224 F = Phe 8 2.985 G = Gly 21 7.836 H = His 3 1.119 I = Ile 17 6.343K = Lys 14 5.224 L = Leu 8 2.985 M = Met 4 1.493 N = Asn 18 6.716 P =Pro 12 4.478 Q = Gln 5 1.866 R = Arg 8 2.985 S = Ser 19 7.090 T = Thr 4014.925 V = Val 27 10.075 W = Trp 2 0.746 Y = Tyr 16 5.970 Z = Glx 00.000 A + G 36 13.433 Non-polar S + T 59 22.015 Polar D + E 30 11.194Acidic D + E + N + Q 53 19.776 H + K + R 25 9.328 Basic D + E + H + K +R 55 20.522 I + L + M + V 56 20.896 Hydrophobic non-aromatic F + W + Y26 9.701 Aromatic

[0137] tIC101

[0138] The following amino acid sequence, numbered for convenience,represents an example of a CrytIC101 insecticidal protein. The aminoacid sequence is represented at SEQ ID NO:4. One nucleotide sequencewhich encodes the tIC101 amino acid sequence is set forth at SEQ IDNO:3, which indicates the particular codons observed in the native B.t.coding sequence. Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe1               5                   10 Tyr Asn Glu Gly Glu Trp Gly GlyPro Glu Pro Tyr         15                  20 Gly Lys Ile Tyr Ala TyrLeu Gln Asn Pro Asp His 25                  30                  35 AsnPhe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys            40                  45 Asp Thr Pro Glu Lys Ser Ser His ThrGln Thr Ile     50                  55                  60 Lys Ile SerSer Pro Thr Gly Gly Pro Ile Asn Gln                65                  70 Met Cys Phe Tyr Gly Asp Val LysGlu Tyr Asp Val         75                  80 Gly Asn Ala Asp Asp ValLeu Ala Tyr Pro Ser Gln 85                  90                  95 LysVal Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu            100                 105 Asn Gly Asp Glu Lys Gly Ser Tyr IleGln Ile Arg     110                 115                 120 Tyr Ser LeuAla Pro Ala                 125

[0139] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 3.

[0140] Molecular weight=14159. Residues=126

[0141] Isoelectric point=4.70 TABLE 3 Amino Acid Composition of tIC101Residue Type Number of Residues Mole Percent In tIC101 Protein A = Ala 53.968 B = Asx 0 0.000 C = Cys 2 1.587 D = Asp 8 6.349 E = Glu 7 5.556 F= Phe 4 3.175 G = Gly 12 9.524 H = His 2 1.587 I = Ile 9 7.143 K = Lys 86.349 L = Leu 4 3.175 M = Met 2 1.587 N = Asn 8 6.349 P = Pro 9 7.143 Q= Gln 6 4.762 R = Arg 2 1.587 S = Ser 9 7.143 T = Thr 10 7.937 V = Val 64.762 W = Trp 3 2.381 Y = Tyr 10 7.937 Z = Glx 0 0.000 A + G 17 13.492Non-polar S + T 19 5.079 Polar D + E 15 11.905 Acidic D + E + N + Q 2923.016 H + K + R 12 9.524 Basic D + E + H + K + R 27 21.429 I + L + M +V 21 16.667 Hydrophobic non-aromatic F + W + Y 17 13.492 Aromatic

[0142] tIC100/tIC101 Fusion with BamHI/NheI (GSGGAS) Linker

[0143] The following amino acid sequence, numbered for convenience,represents an example of a CrytIC100/CrytIC101 insecticidal proteinfusion between CrytIC100 and CrytIC101, CrytIC100 being positioned atthe amino terminus of the fusion, and containing aGly-Ser-Gly-Gly-Ala-Ser (GSGGAS) amino acid sequence linker between thetwo protein sequences. The underlined amino acids at residues numberedfrom position 269 through position 274 indicate the linker sequence inthis novel insecticidal fusion protein. Met Gly Ile Ile Asn Ile Gln AspGlu Ile Asn Asp 1               5                   10 Tyr Met Lys GlyMet Tyr Gly Ala Thr Ser Val Lys         15                  20 Ser ThrTyr Asp Pro Ser Phe Lys Val Phe Asn Glu25                  30                  35 Ser Val Thr Pro Gln Tyr AspVal Ile Pro Thr Glu             40                  45 Pro Val Asn AsnHis Ile Thr Thr Lys Val Ile Asp    50                  55                  60 Asn Pro Gly Thr Ser GluVal Thr Ser Thr Val Thr                 65                  70 Phe ThrTrp Thr Glu Thr Asp Thr Val Thr Ser Ala         75                  80Val Thr Lys Gly Tyr Lys Val Gly Gly Ser Val Ser85                  90                  95 Ser Lys Ala Thr Phe Lys PheAla Phe Val Thr Ser             100                 105 Asp Val Thr ValThr Val Ser Ala Glu Tyr Asn Tyr    110                 115                 120 Ser Thr Thr Glu Thr ThrThr Lys Thr Asp Thr Arg                 125                 130 Thr TrpThr Asp Ser Thr Thr Val Lys Ala Pro Pro         135                 140Arg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr145                 150                 155 Gly Asn Tyr Asn Val Pro ValAsn Val Glu Ser Asp             160                 165 Met Thr Gly ThrLeu Phe Cys Arg Gly Tyr Arg Asp    170                 175                 180 Gly Ala Leu Ile Ala AlaAla Tyr Val Ser Ile Thr                 185                 190 Asp LeuAla Asp Tyr Asn Pro Asn Leu Gly Leu Thr         195                 200Asn Glu Gly Asn Gly Val Ala His Phe Lys Gly Glu205                 210                 215 Gly Tyr Ile Glu Gly Ala GlnGly Leu Arg Ser Tyr             220                 225 Ile Gln Val ThrGlu Tyr Pro Val Asp Asp Asn Gly    230                 235                 240 Arg His Ser Ile Pro LysThr Tyr Ile Ile Lys Gly                 245                 250 Ser LeuAla Pro Asn Val Thr Leu Ile Asn Asp Arg         255                 260Lys Glu Gly Arg Gly Ser Gly Gly Ala  Ser Met Thr265                 270                 275 Val Tyr Asn Val Thr Phe ThrIle Lys Phe Tyr Asn             280                 285 Glu Gly Glu TrpGly Gly Pro Glu Pro Tyr Gly Lys    290                 295                 300 Ile Tyr Ala Tyr Leu GlnAsn Pro Asp His Asn Phe                 305                 310 Glu IleTrp Ser Gln Asp Asn Trp Gly Lys Asp Thr         315                 320Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys Ile325                 330                 335 Ser Ser Pro Thr Gly Gly ProIle Asn Gln Met Cys             340                 345 Phe Tyr Gly AspVal Lys Glu Tyr Asp Val Gly Asn    350                 355                 360 Ala Asp Asp Val Leu AlaTyr Pro Ser Gln Lys Val                 365                 370 Cys SerThr Pro Gly Thr Thr Ile Arg Leu Asn Gly         375                 380Asp Glu Lys Gly Ser Tyr Ile Gln Ile Arg Tyr Ser385                 390                 395 Leu Ala Pro Ala            400

[0144] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 4.

[0145] Molecular weight=43796. Residues=400

[0146] Isoelectric point=4.75 TABLE 4 Amino Acid Composition oftIC100/tIC101 Fusion [BamHI/NheI (GSGGAS) Linker] Residue Type Number ofResidues Mole Percent In tIC100/101 Protein A = Ala 21 5.250 B = Asx 00.000 C = Cys 3 0.750 D = Asp 24 6.000 E = Glu 21 5.250 F = Phe 12 3.000G = Gly 36 9.000 H = His 5 1.250 I = Ile 26 6.500 K = Lys 22 5.500 L =Leu 12 3.000 M = Met 6 1.500 N = Asn 26 6.500 P = Pro 21 5.250 Q = Gln11 2.750 R = Arg 10 2.500 S = Ser 30 7.500 T = Thr 50 12.500 V = Val 338.250 W = Trp 5 1.250 Y = Tyr 26 6.500 Z = Glx 0 0.000 A + G 57 14.250Non-polar S + T 80 20.000 Polar D + E 45 11.250 Acidic D + E + N + Q 8220.500 H + K + R 37 9.250 Basic D + E + H + K + R 82 20.500 I + L + M +V 77 19.250 Hydrophobic non-aromatic F + W + Y 43 10.750 Aromatic

[0147] An insecticidal fusion protein similar to the tIC100/tIC101fusion described in above and in Table 4 was constructed, but the DNAsequence representing the open reading frame encoding tIC101 peptide waspositioned at the 5′ end of the cassette so that the tIC101 peptidewould be positioned at the amino terminal position of the fusionprotein, while the DNA sequence representing the open reading frameencoding the tIC100 peptide was positioned toward the 3′ end of thecassette so that the tIC100 peptide would be positioned at the carboxyterminal position of the fusion protein. The two proteins were alsolinked in frame by a sequence encoding a Gly-Ser-Gly-Gly-Ala-Ser(GSGGAS) linker peptide as described above. The resulting amino acidsequence of the fusion peptide, tIC101/tIC100, was identical in aminoacid composition analysis to the tIC100/tIC101 fusion peptide describedin Table 4, and exhibiting a molecular weight of 43796 Da, comprising400 amino acid residues, and exhibiting a calculated isoelectric pointof 4.75. This fusion peptide was also shown to demonstrate an effectivecoleopteran insect inhibitory bioactivity, in particular in cotton bollweevil bioassay. The amino acid sequence of the tIC101/tIC100 fusionpeptide linked in frame by a GSGGAS linker is shown below, and theunderlined residues at amino acid sequence positions 127-132 representthe GSGGAS linker: Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe1               5                   10 Tyr Asn Glu Gly Glu Trp Gly GlyPro Glu Pro Tyr         15                  20 Gly Lys Ile Tyr Ala TyrLeu Gln Asn Pro Asp His 25                  30                  35 AsnPhe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys            40                  45 Asp Thr Pro Glu Lys Ser Ser His ThrGln Thr Ile     50                  55                  60 Lys Ile SerSer Pro Thr Gly Gly Pro Ile Asn Gln                65                  70 Met Cys Phe Tyr Gly Asp Val LysGlu Tyr Asp Val         75                  80 Gly Asn Ala Asp Asp ValLeu Ala Tyr Pro Ser Gln 85                  90                  95 LysVal Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu            100                 105 Asn Gly Asp Glu Lys Gly Ser Tyr IleGln Ile Arg     110                 115                 120 Tyr Ser LeuAla Pro Ala Gly Ser Gly Gly Ala Ser                125                 130 Met Gly Ile Ile Asn Ile Gln AspGlu Ile Asn Asp         135                 140 Tyr Met Lys Gly Met TyrGly Ala Thr Ser Val Lys 145                 150                 155 SerThr Tyr Asp Pro Ser Phe Lys Val Phe Asn Glu            160                 165 Ser Val Thr Pro Gln Tyr Asp Val IlePro Thr Glu     170                 175                 180 Pro Val AsnAsn His Ile Thr Thr Lys Val Ile Asp                185                 190 Asn Pro Gly Thr Ser Glu Val ThrSer Thr Val Thr         195                 200 Phe Thr Trp Thr Glu ThrAsp Thr Val Thr Ser Ala 205                 210                 215 ValThr Lys Gly Tyr Lys Val Gly Gly Ser Val Ser            220                 225 Ser Lys Ala Thr Phe Lys Phe Ala PheVal Thr Ser     230                 235                 240 Asp Val ThrVal Thr Val Ser Ala Glu Tyr Asn Tyr                245                 250 Ser Thr Thr Glu Thr Thr Thr LysThr Asp Thr Arg         255                 260 Thr Trp Thr Asp Ser ThrThr Val Lys Ala Pro Pro 265                 270                 275 ArgThr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr            280                 285 Gly Asn Tyr Asn Val Pro Val Asn ValGlu Ser Asp     290                 295                 300 Met Thr GlyThr Leu Phe Cys Arg Gly Tyr Arg Asp                305                 310 Gly Ala Leu Ile Ala Ala Ala TyrVal Ser Ile Thr         315                 320 Asp Leu Ala Asp Tyr AsnPro Asn Leu Gly Leu Thr 325                 330                 335 AsnGlu Gly Asn Gly Val Ala His Phe Lys Gly Glu            340                 345 Gly Tyr Ile Glu Gly Ala Gln Gly LeuArg Ser Tyr     350                 355                 360 Ile Gln ValThr Glu Tyr Pro Val Asp Asp Asn Gly                365                 370 Arg His Ser Ile Pro Lys Thr TyrIle Ile Lys Gly         375                 380 Ser Leu Ala Pro Asn ValThr Leu Ile Asn Asp Arg 385                 390                 395 LysGlu Gly Arg             400

[0148] tIC101/tIC100 Fusion with Gly-Gly Linker

[0149] The following amino acid sequence, numbered for convenience,represents an example of a CrytIC101/CrytIC100 insecticidal proteinfusion between CrytIC101 and CrytIC100, CrytIC101 being positioned atthe amino terminus of the fusion, and containing a Gly-Gly (GG)dipeptide linker between the two protein sequences. The underlined aminoacids at residues numbered from position 127 through position 128indicate the linker sequence in this novel insecticidal fusion protein.Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe1               5                   10 Tyr Asn Glu Gly Glu Trp Gly GlyPro Glu Pro Tyr         15                  20 Gly Lys Ile Tyr Ala TyrLeu Gln Asn Pro Asp His 25                  30                  35 AsnPhe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys            40                  45 Asp Thr Pro Glu Lys Ser Ser His ThrGln Thr Ile     50                  55                  60 Lys Ile SerSer Pro Thr Gly Gly Pro Ile Asn Gln                65                  70 Met Cys Phe Tyr Gly Asp Val LysGlu Tyr Asp Val         75                  80 Gly Asn Ala Asp Asp ValLeu Ala Tyr Pro Ser Gln 85                  90                  95 LysVal Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu            100                 105 Asn Gly Asp Glu Lys Gly Ser Tyr IleGln Ile Arg     110                 115                 120 Tyr Ser LeuAla Pro Ala Gly Gly Met Gly Ile Ile                125                 130 Asn Ile Gln Asp Glu Ile Asn AspTyr Met Lys Gly         135                 140 Met Tyr Gly Ala Thr SerVal Lys Ser Thr Tyr Asp 145                 150                 155 ProSer Phe Lys Val Phe Asn Glu Ser Val Thr Pro            160                 165 Gln Tyr Asp Val Ile Pro Thr Glu ProVal Asn Asn     170                 175                 180 His Ile ThrThr Lys Val Ile Asp Asn Pro Gly Thr                185                 190 Ser Glu Val Thr Ser Thr Val ThrPhe Thr Trp Thr         195                 200 Glu Thr Asp Thr Val ThrSer Ala Val Thr Lys Gly 205                 210                 215 TyrLys Val Gly Gly Ser Val Ser Ser Lys Ala Thr            220                 225 Phe Lys Phe Ala Phe Val Thr Ser AspVal Thr Val     230                 235         240 Thr Val Ser Ala GluTyr Asn Tyr Ser Thr Thr Glu                 245                 250 ThrThr Thr Lys Thr Asp Thr Arg Thr Trp Thr Asp        255                 260 Ser Thr Thr Val Lys Ala Pro Pro Arg ThrAsn Val 265                 270                 275 Glu Val Ala Tyr IleIle Gln Thr Gly Asn Tyr Asn             280                 285 Val ProVal Asn Val Glu Ser Asp Met Thr Gly Thr    290                 295                 300 Leu Phe Cys Arg Gly TyrArg Asp Gly Ala Leu Ile                 305                 310 Ala AlaAla Tyr Val Ser Ile Thr Asp Leu Ala Asp         315                 320Tyr Asn Pro Asn Leu Gly Leu Thr Asn Glu Gly Asn325                 330                 335 Gly Val Ala His Phe Lys GlyGlu Gly Tyr Ile Glu             340                 345 Gly Ala Gln GlyLeu Arg Ser Tyr Ile Gln Val Thr    350                 355                 360 Glu Tyr Pro Val Asp AspAsn Gly Arg His Ser Ile                 365                 370 Pro LysThr Tyr Ile Ile Lys Gly Ser Leu Ala Pro         375                 380Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly Arg385                 390                 395

[0150] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 5.

[0151] Molecular weight=43494. Residues=396

[0152] Isoelectric point=4.75 TABLE 5 Amino Acid Composition oftIC100/101 Fusion [Gly-Gly Linker] Residue Type Number of Residues MolePercent In tIC100 Protein A = Ala 20 5.051 B = Asx 0 0.000 C = Cys 30.758 D = Asp 24 6.061 E = Glu 21 5.303 F = Phe 12 3.030 G = Gly 358.838 H = His 5 1.263 I = Ile 26 6.566 K = Lys 22 5.556 L = Leu 12 3.030M = Met 6 1.515 N = Asn 26 6.566 P = Pro 21 5.303 Q = Gln 11 2.778 R =Arg 10 2.525 S = Ser 28 7.071 T = Thr 50 12.626 V = Val 33 8.333 W = Trp5 1.263 Y = Tyr 26 6.566 Z = Glx 0 0.000 A + G 55 13.889 Non-polar S + T78 19.697 Polar D + E 45 11.364 Acidic D + E + N + Q 82 0.707 H + K + R37 9.343 Basic D + E + H + K + R 82 20.707 I + L + M + V 77 19.444Hydrophobic non-aromatic F + W + Y 43 10.859 Aromatic

[0153] CryET33/CryET34 Fusion with BamHI/NheI (GSGGAS) Linker

[0154] The following amino acid sequence, numbered for convenience,represents an example of a CryET33/CryET34 insecticidal protein fusionbetween CryET33 and CryET34 and containing a Gly-Ser-Gly-Gly-Ala-Ser(GSGGAS) amino acid sequence linker between the two protein sequences.The underlined amino acids at residues numbered from position 268through position 273 indicate the linker sequence in this novelinsecticidal fusion protein. Met Gly Ile Ile Asn Ile Gln Asp Glu Ile AsnAsn 1               5                   10 Tyr Met Lys Glu Val Tyr GlyAla Thr Thr Val Lys         15                  20 Ser Thr Tyr Asp ProSer Phe Lys Val Phe Asn Glu 25                  30                  35Ser Val Thr Pro Gln Phe Thr Glu Ile Pro Thr Glu            40                  45 Pro Val Asn Asn Gln Leu Thr Thr LysArg Val Asp     50                  55                  60 Asn Thr GlySer Tyr Pro Val Glu Ser Thr Val Ser                65                  70 Phe Thr Trp Thr Glu Thr His ThrGlu Thr Ser Ala         75                  80 Val Thr Glu Gly Val LysAla Gly Thr Ser Ile Ser 85                  90                  95 ThrLys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser            100                 105 Asp Val Thr Leu Thr Val Ser Ala GluTyr Asn Tyr     110                 115                 120 Ser Thr ThrAsn Thr Thr Thr Thr Thr Glu Thr His                125                 130 Thr Trp Ser Asp Ser Thr Lys ValThr Ile Pro Pro         135                 140 Lys Thr Tyr Val Glu AlaAla Tyr Ile Ile Gln Asn 145                 150                 155 GlyThr Tyr Asn Val Pro Val Asn Val Glu Cys Asp            160                 165 Met Ser Gly Thr Leu Phe Cys Arg GlyTyr Arg Asp     170                 175                 180 Gly Ala LeuIle Ala Ala Val Tyr Val Ser Val Ala                185                 190 Asp Leu Ala Asp Tyr Asn Pro AsnLeu Asn Leu Thr         195                 200 Asn Lys Gly Asp Gly IleAla His Phe Lys Gly Ser 205                 210                 215 GlyPhe Ile Glu Gly Ala Gln Gly Leu Arg Ser Ile            220                 225 Ile Gln Val Thr Glu Tyr Pro Leu AspAsp Asn Lys     230                 235                 240 Gly Arg SerThr Pro Ile Thr Tyr Leu Ile Asn Gly                245                 250 Ser Leu Ala Pro Asn Val Thr LeuLys Asn Ser Asn         255                 260              265 Ile LysPhe Gly Ser Gly Gly Ala Ser Met Thr Val           270                          275 Tyr Asn Ala Thr Phe Thr IleAsn Phe Tyr Asn Glu                  280                 285 Gly Glu TrpGly Gly Pro Glu Pro Tyr Gly Tyr Ile    290                 295                 300 Lys Ala Tyr Leu Thr AsnPro Asp His Asp Phe Glu                 305                 310 Ile TrpLys Gln Asp Asp Trp Gly Lys Ser Thr Pro         315                 320Glu Arg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser325                 330                 335 Ser Asp Thr Gly Ser Pro IleAsn Gln Met Cys Phe             340                 345 Tyr Gly Asp ValLys Glu Tyr Asp Val Gly Asn Ala    350                 355                 360 Asp Asp Ile Leu Ala TyrPro Ser Gln Lys Val Cys                 365                 370 Ser ThrPro Gly Val Thr Val Arg Leu Asp Gly Asp         375                 380Glu Lys Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu385                 390                 395 Thr Pro Ala

[0155] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 6.

[0156] Molecular weight=43792. Residues=399

[0157] Isoelectric point=4.53 TABLE 6 Amino Acid Composition ofCryET33/CryET34 Fusion [BamHI/NheI (GSGGAS) Linker Residue Type Numberof Residues Mole Percent In CryET33/34 Protein A = Ala 20 5.013 B = Asx0 0.000 C = Cys 4 1.003 D = Asp 23 5.764 E = Glu 22 5.514 F = Phe 153.759 G = Gly 32 8.020 H = His 4 1.003 I = Ile 25 6.266 K = Lys 22 5.514L = Leu 16 4.010 M = Met 5 1.253 N = Asn 28 7.018 P = Pro 20 5.013 Q =Gln 11 2.757 R = Arg 7 1.754 S = Ser 33 8.271 T = Thr 52 13.033 V = Val30 7.519 W = Trp 5 1.253 Y = Tyr 25 6.266 Z = Glx 0 0.000 A + G 5213.033 Non-polar S + T 85 21.303 Polar D + E 45 11.278 Acidic D + E +N + Q 84 21.053 H + K + R 33 8.271 Basic D + E + H + K + R 78 19.549 I +L + M + V 76 19.048 Hydrophobic non-aromatic F + W + Y 45 11.278Aromatic

[0158] CryET33/CryET34 Fusion with (GGGS)₃ Linker

[0159] The following amino acid sequence, numbered for convenience,represents an example of a CryET33/CryET34 insecticidal protein fusionbetween CryET33 and CryET34 and containing aGly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ala-Ser (GGGS)₃amino acid sequence linker between the two protein sequences. Theunderlined amino acids at residues numbered from position 268 throughposition 283 indicate the linker sequence in this novel insecticidalfusion protein. Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn1               5                   10 Tyr Met Lys Glu Val Tyr Gly AlaThr Thr Val Lys         15                  20 Ser Thr Tyr Asp Pro SerPhe Lys Val Phe Asn Glu 25                  30                  35 SerVal Thr Pro Gln Phe Thr Glu Ile Pro Thr Glu            40                  45 Pro Val Asn Asn Gln Leu Thr Thr LysArg Val Asp     50                  55                  60 Asn Thr GlySer Tyr Pro Val Glu Ser Thr Val Ser                65                  70 Phe Thr Trp Thr Glu Thr His ThrGlu Thr Ser Ala         75                  80 Val Thr Glu Gly Val LysAla Gly Thr Ser Ile Ser 85                  90                  95 ThrLys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser            100                 105 Asp Val Thr Leu Thr Val Ser Ala GluTyr Asn Tyr     110                 115                 120 Ser Thr ThrAsn Thr Thr Thr Thr Thr Glu Thr His                125                 130 Thr Trp Ser Asp Ser Thr Lys ValThr Ile Pro Pro         135                 140 Lys Thr Tyr Val Glu AlaAla Tyr Ile Ile Gln Asn 145                 150                 155 GlyThr Tyr Asn Val Pro Val Asn Val Glu Cys Asp            160                 165 Met Ser Gly Thr Leu Phe Cys Arg GlyTyr Arg Asp     170                 175                 180 Gly Ala LeuIle Ala Ala Val Tyr Val Ser Val Ala                185                 190 Asp Leu Ala Asp Tyr Asn Pro AsnLeu Asn Leu Thr         195                 200 Asn Lys Gly Asp Gly IleAla His Phe Lys Gly Ser 205                 210                 215 GlyPhe Ile Glu Gly Ala Gln Gly Leu Arg Ser Val            220                 225 Ile Gln Val Thr Glu Tyr Pro Leu AspAsp Asn Lys     230                 235                 240 Gly Arg SerThr Pro Ile Thr Tyr Leu Ile Asn Gly                245                 250 Ser Leu Ala Pro Asn Val Thr LeuLys Asn Ser Asn         255                 260 Ile Lys PheGly Ser Gly Gly Gly Ser Gly Gly Gly265                 270                 275Ser Gly Gly Gly Ser Ala Ser Met Thr Val Tyr Asn            280                 285 Ala Thr Phe Thr Ile Asn Phe Tyr AsnGlu Gly Glu     290                 295                 300 Trp Gly GlyPro Glu Pro Tyr Gly Tyr Ile Lys Ala                305                 310 Tyr Leu Thr Asn Pro Asp His AspPhe Glu Ile Trp         315                 320 Lys Gln Asp Asp Trp GlyLys Ser Thr Pro Glu Arg 325                 330                 335 SerThr Tyr Thr Gln Thr Ile Lys Ile Ser Ser Asp            340                 345 Thr Gly Ser Pro Ile Asn Gln Met CysPhe Tyr Gly     350                 355                 360 Asp Val LysGlu Tyr Asp Val Gly Asn Ala Asp Asp                365                 370 Ile Leu Ala Tyr Pro Ser Gln LysVal Cys Ser Thr         375                 380 Pro Gly Val Thr Val ArgLeu Asp Gly Asp Glu Lys 385                 390                 395 GlySer Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro             400 Ala            405

[0160] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 7.

[0161] Molecular weight=44453. Residues=409

[0162] Isoelectric point=4.53 TABLE 7 Amino Acid Composition ofCryET33/CryET34 [(GGGS)₃ Linker] Residue Type Number of Residues MolePercent In CryET33/34 Protein A = ALA 20 4.890 B = Asx 0 0.000 C = Cys 40.978 D = Asp 23 5.623 E = Glu 22 5.379 F = Phe 15 3.667 G = Gly 399.535 H = His 4 0.978 I = Ile 25 6.112 K = Lys 22 5.379 L = Leu 16 3.912M = Met 5 1.222 N = Asn 28 6.846 P = Pro 20 4.890 Q = Gln 11 2.689 R =Arg 7 1.711 S = Ser 36 8.802 T = Thr 52 12.714 V = Val 30 7.335 W = Trp5 1.222 Y = Tyr 25 6.112 Z = Glx 0 0.000 A + G 59 14.425 Non-polar S + T88 21.516 Polar D + E 45 11.002 Acidic D + E + N + Q 84 20.538 H + K + R33 8.068 Basic D + E + H + K + R 78 19.071 I + L + M + V 76 18.582Hydrophobic non-aromatic F + W + Y 45 11.002 Aromatic

[0163] CryET33/CryET34 Fusion with Lysine Oxidase (PALLKEAPRAEEELPP)Linker

[0164] The following amino acid sequence, numbered for convenience,represents an example of a CryET33/CryET34 insecticidal protein fusionbetween CryET33 and CryET34 and containing a lysine oxidase amino acidsequence linker between the two protein sequences. The underlined aminoacids at residues numbered from position 268 through position 287indicate the lysine oxidase linker sequence in this novel insecticidalfusion protein. Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn1               5                   10 Tyr Met Lys Glu Val Tyr Gly AlaThr Thr Val Lys         15                  20 Ser Thr Tyr Asp Pro SerPhe Lys Val Phe Asn Glu 25                  30                  35 SerVal Thr Pro Gln Phe Thr Glu Ile Pro Thr Glu            40                  45 Pro Val Asn Asn Gln Leu Thr Thr LysArg Val Asp     50                  55                  60 Asn Thr GlySer Tyr Pro Val Glu Ser Thr Val Ser                65                  70 Phe Thr Trp Thr Glu Thr His ThrGlu Thr Ser Ala         75                  80 Val Thr Glu Gly Val LysAla Gly Thr Ser Ile Ser 85                  90                  95 ThrLys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser 100                 105 AspVal Thr Leu Thr Val Ser Ala Glu Tyr Asn Tyr    110                 115                 120 Ser Thr Thr Asn Thr ThrThr Thr Thr Glu Thr His                 125                 130 Thr TrpSer Asp Ser Thr Lys Val Thr Ile Pro Pro         135                 140Lys Thr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn145                 150                 155 Gly Thr Tyr Asn Val Pro ValAsn Val Glu Cys Asp             160                 165 Met Ser Gly ThrLeu Phe Cys Arg Gly Tyr Arg Asp    170                 175                 180 Gly Ala Leu Ile Ala AlaVal Tyr Val Ser Val Ala                 185                 190 Asp LeuAla Asp Tyr Asn Pro Asn Leu Asn Leu Thr         195                 200Asn Lys Gly Asp Gly Ile Ala His Phe Lys Gly Ser205                 210                 215 Gly Phe Ile Glu Gly Ala GlnGly Leu Arg Ser Val             220                 225 Ile Gln Val ThrGlu Tyr Pro Leu Asp Asp Asn Lys    230                 235                 240 Gly Arg Ser Thr Pro IleThr Tyr Leu Ile Asn Gly                 245                 250 Ser LeuAla Pro Asn Val Thr Leu Lys Asn Ser Asn        255                 260              265 Ile Lys PheGly Ser Pro Ala Leu Leu Lys Glu Ala           270                          275Pro Arg Ala Glu Glu Glu Leu Pro Pro Ala Ser Met            280                 285 Thr Val Tyr Asn Ala Thr Phe Thr IleAsn Phe Tyr     290                 295                 300 Asn Glu GlyGlu Trp Gly Gly Pro Glu Pro Tyr Gly                305                 310 Tyr Ile Lys Ala Tyr Leu Thr AsnPro Asp His Asp         315                 320 Phe Glu Ile Trp Lys GlnAsp Asp Trp Gly Lys Ser 325                 330                 335 ThrPro Glu Arg Ser Thr Tyr Thr Gln Thr Ile Lys            340                 345 Ile Ser Ser Asp Thr Gly Ser Pro IleAsn Gln Met     350                 355                 360 Cys Phe TyrGly Asp Val Lys Glu Tyr Asp Val Gly                365                 370 Asn Ala Asp Asp Ile Leu Ala TyrPro Ser Gln Lys         375                 380 Val Cys Ser Thr Pro GlyVal Thr Val Arg Leu Asp 385                 390                 395 GlyAsp Glu Lys Gly Ser Tyr Val Thr Ile Lys Tyr            400                 405 Ser Leu Thr Pro Ala     410

[0165] The resulting protein is calculated to comprise the followingcomposition, including the amino acid sequence residues, number of eachamino acid residue, and mole percent of each combination of residues ofa particular species as set forth in Table 8.

[0166] Molecular weight=45420. Residues=413

[0167] Isoelectric point=4.51 TABLE 8 Amino Acid CompositionCryET33/ET34 Fusion [lysine oxidase (PALLKEAPRAEEELPP) linker] ResidueType Number of Residues Mole Percent In CryET33/34 Protein A = Ala 235.569 B = Asx 0 0.000 C = Cys 4 0.969 D = Asp 23 5.569 E = Glu 26 6.295F = Phe 15 3.632 G = Gly 30 7.264 H = His 4 0.969 I = Ile 25 6.053 K =Lys 23 5.569 L = Leu 19 4.600 M = Met 5 1.211 N = Asn 28 6.780 P = Pro24 5.811 Q = Gln 11 2.663 R = Arg 8 1.937 S = Ser 33 7.990 T = Thr 5212.591 V = Val 30 7.264 W = Trp 5 1.211 Y = Tyr 25 6.053 Z = Glx 0 0.000A + G 53 12.833 Non-polar S + T 85 20.581 Polar D + E 49 11.864 AcidicD + E + N + Q 88 21.308 H + K + R 35 8.475 Basic D + E + H + K + R 8420.339 I + L + M + V 79 19.128 Hydrophobic non-aromatic F + W + Y 4510.896 Aromatic

[0168] Nomenclature of the Novel Proteins

[0169] The inventors have arbitrarily assigned the designations tIC100and tIC101 to the novel proteins, and tIC100 and tIC101 to the novelnucleic acid sequences encoding the respective polypeptides. Formalassignment of gene and protein designations based on the revisednomenclature of crystal protein endotoxins may be assigned by acommittee on the nomenclature of B. thuringiensis, formed tosystematically classify B. thuringiensis crystal proteins. The inventorscontemplate that the official nomenclature assigned to these sequenceswill supercede the arbitrarily assigned designations of the presentinvention.

[0170] Transformed Host Cells and Transgenic Plants

[0171] Methods and compositions for transforming a bacterium, a yeastcell, a plant cell, or an entire plant with one or more expressionvectors comprising a crystal protein-encoding gene sequence are furtheraspects of this disclosure. A transgenic bacterium, yeast cell, plantcell or plant derived from such a transformation process or the progenyand seeds from such a transgenic plant are also further embodiments ofthe invention.

[0172] Means for transforming bacteria and yeast cells are well known inthe art. Typically, means of transformation are similar to those wellknown means used to transform other bacteria or yeast such as E coli orSaccharomyces cerevisiae. Methods for DNA transformation of plant cellsinclude Agrobacterium-mediated plant transformation, protoplasttransformation, gene transfer into pollen, injection into reproductiveorgans, injection into immature embryos and particle bombardment. Eachof these methods has distinct advantages and disadvantages. Thus, oneparticular method of introducing genes into a particular plant strainmay not necessarily be the most effective for another plant strain, butit is well known which methods are useful for a particular plant strain.

[0173] There are many methods for introducing transforming DNA sequencesinto cells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

[0174] Technology for introduction of DNA into cells is well-known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, 1973; Zatloukal et al., 1992); (2) physical methods such asmicroinjection (Capecchi, 1980), electroporation (Wong and Neumann,1982; Fromm et al., 1985; U.S. Pat. No. 5,384,253) and the gene gun(Johnston and Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp,1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 1988b); and (4)receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al.,1992).

[0175] Electroporation

[0176] The application of brief, high-voltage electric pulses to avariety of animal and plant cells leads to the formation ofnanometer-sized pores in the plasma membrane. DNA is taken directly intothe cell cytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of clones genes and for establishment of celllines that carry integrated copies of the gene of interest.Electroporation, in contrast to calcium phosphate-mediated transfectionand

[0177] protoplast fusion, frequently gives rise to cell lines that carryone, or at most a few, integrated copies of the foreign DNA.

[0178] The introduction of DNA by means of electroporation is well-knownto those of skill in the art. In this method, certain cellwall-degrading enzymes, such as pectin degrading enzymes, are employedto render the target recipient cells more susceptible to transformationby electroporation than untreated cells. Alternatively, recipient cellsare made more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

[0179] Microprojectile Bombardment

[0180] A further advantageous method for delivering transforming DNAsequences to plant cells is microprojectile bombardment. In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

[0181] An advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly stably transforming monocots,is that neither the isolation of protoplasts (Cristou et al., 1988) northe susceptibility to Agrobacterium infection is required. Anillustrative embodiment of a method for delivering DNA into maize cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withcorn cells cultured in suspension. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing damage inflicted on the recipient cells by projectiles that aretoo large.

[0182] For the bombardment, cells in suspension are preferablyconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the macroprojectile stopping plate. If desired, one or morescreens are also positioned between the acceleration device and thecells to be bombarded. Through the use of techniques set forth hereinone may obtain up to 1000 or more foci of cells transiently expressing amarker gene. The number of cells in a focus which express the exogenousgene product 48 hours post-bombardment often range from 1 to 10 andaverage 1 to 3.

[0183] In bombardment transformation, one may optimize theprebombardment culturing conditions and the bombardment parameters toyield the maximum numbers of stable transformants. Both the physical andbiological parameters for bombardment are important in this technology.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmids. It is believed that pre-bombardment manipulationsare especially important for successful transformation of immatureembryos.

[0184] Accordingly, it is contemplated that one may wish to adjustvarious of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

[0185] Agrobacterium-Mediated Transfer

[0186] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

[0187] Modem Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacteriun-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

[0188] Agrobacterium-mediated transformation of leaf disks and othertissues such as cotyledons and hypocotyls appears to be limited toplants that Agrobacterium naturally infects. Agrobacterium-mediatedtransformation is most efficient in dicotyledonous plants. Few monocotsappear to be natural hosts for Agrobacterium, although transgenic plantshave been produced in asparagus using Agrobacterium vectors as described(Bytebier et al., 1987). Therefore, commercially important cereal grainssuch as rice, corn, and wheat must usually be transformed usingalternative methods. However, as mentioned above, the transformation ofasparagus using Agrobacterium can also be achieved (see, for example,Bytebier et al., 1987). Recently, Jinjiang et al. (U.S. Pat. No.6,037,522; 2000) disclosed a method for efficient Agrobacterium mediatedtransformation of monocots.

[0189] A transgenic plant regenerated from Agrobacterium mediatedtransformation methods typically contains a single simple insert on onechromosome. Such transgenic plants can be referred to as beingheterozygous for the added insert, and for coding sequences containedwithin the insert. However, inasmuch as use of the word “heterozygous”usually implies the presence of a complementary sequence at the samelocus of the second chromosome of a pair of chromosomes, and there is nosuch sequence in a plant containing a single simple insert, it isbelieved that a more accurate name for such a plant is an independentsegregant, because the added, exogenous single simple insert segregatesindependently during mitosis and meiosis.

[0190] More preferred is a transgenic plant that is homozygous for theadded structural coding sequence; i.e., a transgenic plant that containstwo or more coding sequences artificially introduced using transgenicmethods, for example by Agrobacterium mediated transformation, onecoding sequence at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfmg) an independent segregant transgenic plant that contains asingle artificially introduced coding sequence, germinating some of theseed produced and analyzing the resulting plants produced for enhancedcarboxylase activity relative to a control (native, non-transgenic) oran independent segregant transgenic plant.

[0191] It is to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous coding sequences. Selfing of appropriateprogeny can produce plants that are homozygous for both artificiallyintroduced simple insert sequences that encode a polypeptides ofinterest. Back-crossing to a parental plant and out-crossing with anon-transgenic plant are also contemplated.

[0192] Other Transformation Methods

[0193] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

[0194] Application of these systems to different plant strains dependsupon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986;Yamada et al., 1986; Abdullah et al., 1986).

[0195] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,1988). In addition, “particle gun” or high-velocity microprojectiletechnology can be utilized. (Vasil, 1992).

[0196] Using that latter technology, DNA is carried through the cellwall and into the cytoplasm on the surface of small metal particles asdescribed (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988).The metal particles penetrate through several layers of cells and thusallow the transformation of cells within tissue explants.

[0197] Methods for Producing Insect-Resistant Transgenic Plants

[0198] By transforming a suitable host cell, such as a plant cell, forexample with a sequence encoding a CryET33/CryET34 fusion peptide or atIC100 and/or tIC101 peptide(s), the expression of the encoded crystalfusion protein (i.e., a bacterial crystal protein or polypeptide havingcoleopteran-inhibitory activity) can result in the formation ofinsect-resistant plants.

[0199] By way of example, one may utilize an expression vectorcontaining a coding region for a B. thuringiensis crystal protein and anappropriate selectable marker to transform a suspension of embryonicplant cells, such as wheat or corn cells using a method such as particlebombardment (Maddock et al., 1991; Vasil et al., 1992) to deliver theDNA coated on microprojectiles into the recipient cells. Transgenicplants are then regenerated from transformed embryonic calli thatexpress the insect inhibitory proteins.

[0200] The formation of transgenic plants may also be accomplished usingother methods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983; Jinjiang etal., 2000). Alternatively, DNA can be introduced into plants by directDNA transfer into pollen (Zhou et al., 1983; Hess, 1987; Luo et al.,1988), by injection of the DNA into reproductive organs of a plant (Penaet al., 1987), or by direct injection of DNA into the cells of immatureembryos followed by the rehydration of desiccated embryos (Neuhaus etal., 1987; Benbrook et al., 1986).

[0201] The regeneration, development, and cultivation of plants fromsingle plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, 1988). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

[0202] The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

[0203] This procedure typically produces shoots within two to fourmonths and those shoots are then transferred to an appropriateroot-inducing medium containing the selective agent and an antibiotic toprevent bacterial growth. Shoots that rooted in the presence of theselective agent to form plantlets are then transplanted to soil or othermedia to allow the production of roots. These procedures vary dependingupon the particular plant strain employed, such variations being wellknown in the art.

[0204] Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

[0205] A transgenic plant of this invention thus has an increased amountof a coding region (e.g., a cry gene) that encodes the Cry polypeptideof interest. A preferred transgenic plant is an independent segregantand can transmit that gene and its activity to its progeny. A morepreferred transgenic plant is homozygous for that gene, and transmitsthat gene to all of its offspring on sexual mating. Seed from atransgenic plant may be grown in the field or greenhouse, and resultingsexually mature transgenic plants are self-pollinated to generate truebreeding plants. The progeny from these plants become true breedinglines that are evaluated for, by way of example, increased insectinhibitory capacity against coleopteran insects, preferably in thefield, under a range of environmental conditions. The inventorscontemplate that the present invention will find particular utility inthe creation of transgenic plants of commercial interest includingvarious cotton, potato, soybean, canola, tomato, turf grasses, wheat,corn, rice, barley, oats, a variety of ornamental plants and vegetables,as well as a number of nut- and fruit-bearing trees and plants.

[0206] Illustrative Embodiments

[0207] This application discloses novel insecticidal proteins isolatablefrom Bacillus thuringiensis strains of bacterium, and in particular,insecticidal proteins exhibiting Coleopteran insecticidal activity. Forthe purposes of this disclosure, the phrase “insect inhibitory” shouldbe correlated with the word “insecticidal”, and these words and phrasesare meant to be used interchangeably herein throughout. A compositioncomprising one or more of the peptides disclosed herein is considered tobe insecticidal, and the term insecticidal, and by analogy “insectinhibitory”, is intended to be defined as a protein which, uponingestion into the digestive system of a target insect, causes morbidityand mortality, in that the target insect, having consumed a quantity ofthe protein is discouraged from eating further, and preferably thetarget insect's growth is stunted or reduced, and more preferably thetarget insect is subjected to drying, desiccation, and death upon eatingan amount of a substance containing the insecticidal protein in anamount sufficient to cause growth inhibition, feeding inhibition,rejection of a substance containing the protein as a food source, andpreferably death.

[0208] An exemplary insecticidal composition comprises a sample whichcontains, in approximately equimolar concentrations, both of theproteins herein defined as CrytIC100 and CrytIC101, alternatively knownas tIC100 and tIC101. These proteins have been identified as beingexpressible from a nucleotide sequence obtained from Bacillusthuringiensis strain EG9328. In the course of identifying Bacillusthuringiensis strains which exhibit Coleopteran activity, sequencescomplementary to the binary toxin composition CryET33 and CryET34 wereused as probes and primers for hybridizing to and/or amplifyingsequences from B.t. strains exhibiting Coleopteran insecticidalactivity. As a result of this hybridization and thermal amplificationanalysis, several strains were identified as containing DNA sequenceswhich contain sequences exhibiting substantial homology to cryET33 andcryET34 DNA sequences and which provided a template for the thermalamplification reaction which produced one or more bands separable uponagarose gel electrophoresis and ethidium bromide staining similar insize to the operon sequence encoding the CryET33 and CryET34 proteins.It was suspected that these bands all encoded the ET33 and ET34 proteinsor homologs thereof. It was surprising that one particular cloneisolated from this amplification analysis failed to produce any crystalmorphology when transformed into an acrystalliferous strain of B.t.Furthermore, DNA sequence analysis of this particular clone resulted inthe identification of a sequence which may have, in evolutionary terms,previously encoded at least two proteins similar but not identical toCryET33 and CryET34. This sequence, and the cryptic operon containedwithin the sequence, isolated from B.t. strain EG9328, is set forthherein in SEQ ID NO:27. While it is impossible to predict whetherthroughout evolutionary time there was one or more bases added to thesequence to disrupt the coding sequence of CrytIC100, or whether therewere one or more bases that were removed from the sequence to disruptthe coding sequence of CrytIC100, or even whether there was ever aCrytIC100 protein ever produced by a Bacillus thuringiensis in nature,the fact remains that removing one of the cytosine residues fromnucleotide position 84 through 88 within the cryptic sequence as setforth in SEQ ID NO:27 causes the reading frame from nucleotide position1 through nucleotide position 804 to shift such that a single openreading frame is created which allows this “corrected” sequence toencode the peptide herein described as tIC100. When expressed along withtIC101, or when tIC100 and tIC101 are present in a sample inapproximately equimolar ratios, the combination of the two proteinsresults in an insecticidal composition, in particular when provided inan orally acceptable diet to a Coleopteran target insect. In particular,the Coleopteran target insect most prevalently affected by the tIC100and tIC101 composition is a boll weevil insect, which is prevalentlyfound as a pest among cotton crops in the new world, i.e., in NorthAmerica, Mexico, Central and South America, and Australia. It was alsofound by the inventors herein that fusions between these two proteinsexhibited insecticidal activity when tested against the boll weevil, andthat it was irrelevant whether the protein fusion contained CrytIC100 orCrytIC101 at the amino terminus of the fusion protein. It was alsodetermined that it was irrelevant as to which proteolyticallysusceptible amino acid sequence linker was present and in frame betweenthe two CrytIC proteins, so long as the linker sequence was capable ofbeing cleaved when the fusion protein was ingested in an orallyacceptable medium by the boll weevil.

[0209] The orally acceptable insect diet or orally administrable dietinto which the insecticidal proteins of the present invention are to beincorporated are well known in the art as described herein. These can beany composition which can be orally ingested by the target insect pesttaking the form for example, when the proteins or fusions of the presentinvention are expressed from within a host cell such as a plant, fungal,or bacterial cell, consisting of a cell extract, a cell suspension, acell homogenate, a cell lysate, a cell supernatant, a cell filtrate, ora cell pellet. In addition, the composition containing the insecticidalprotein(s) of the present invention can be formulated into a powder, adust, a pellet, a granule, a spray, an emulsion, a colloid, or asolution, any of which can be topically applied to a substrate which isor can become an orally ingestible, orally acceptable, or an orallyadministrable diet for a target insect pest. The formulation can beprepared in a number of ways well known in the art, including but not tobe limited to dessication, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration. In any suchorally acceptable, orally administrable, or orally ingestible dietintended for consumption by a target insect pest, the protein of thepresent invention should at least be present in a concentration fromabout 0.001% of the total weight of the composition to about 99% of theweight of the composition.

[0210] In view of the nature of the target pest shown herein to besusceptible to the compositions disclosed herein, it is intended thatnucleotide sequences be synthesized for expression of the proteinaceousagents of the present invention in plant cells, and in particular incotton plant cells. It is well known that Bacillus thuringiensis DNAsequences encoding insecticidal proteins are not preferred forexpression of the proteins encoded thereby in plants. Instead, it hasbeen demonstrated time and again that the preferred DNA sequences forexpression in plants should be artificially synthesized in order tomaximize the levels of expression of the insecticidal proteins inplants. Therefore, it has previously been demonstrated that multiple DNAsequences, because of the redundancy of the genetic code, can encode thesame or a substantially identical protein encoded by the native DNAsequence, i.e. “native” intended to mean “derived as found in nature, oras found in the genome of Bacillus thuringiensis, or in this case,because the coding sequence derived from a plasmid naturally occurringwithin a particular strain of Bacillus thuringiensis”. Therefore, theprior art teachings indicating which codons to use when preparing aparticular nucleotide sequence for expression of a Bt toxin in plantshave been extensively referred to and those disclosures, well known inthe art, are intended to be within the scope of this invention.

EXAMPLES

[0211] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Construction of CryET33/CryET34Insect Inhibitory FusionProtein

[0212] This example illustrates the construction of a DNA sequenceencoding a CryET33 and CryET34 insect inhibitory fusion protein.

[0213] CryET33 and CryET34 peptides and nucleic acid sequences encodingthese novel peptides have been disclosed previously, at least in U.S.Pat. No. 6,063,756. In order to determine whether a CryET33/CryET34fusion can be expressed as a single protein and retain bioactivityagainst boll weevil, a CryET33/CryET34 fusion was constructed based onthe wild-type Bacillus thuringiensis sequences encoding the CryET33 andCryET34 peptides. An expression construct in pMON47407, a Bacillusthuringiensis universal expression vector, was constructed in which theCryET33 coding sequence was downstream of and adjacent to a Bacillusthuringiensis sporulation specific promoter at the 5′-end of theconstruct, and the CryET34 coding sequence was positioned downstream ofand adjacent to the CryET33 coding sequence at the 3′-end of thecassette, mimicking the natural orientation within the native B.t.cryET33 and cryET34 operon. A BamHI/NheI linker sequence encoding theamino acid sequence represented by Gly-Ser-Gly-Gly-Ala-Ser (GSGGAS) wasintroduced in frame between the CryET33 and CryET34 coding sequences toallow for protein flexibility as well as providing a convenientrestriction site sequence for introducing other linkers if necessary(see FIG. 1). The sequence encoding the CryET33/CryET34 fusion wasconstructed using overlapping thermal amplification mutagenesis, andincorporated an SpeI site at the 5′-end and an XhoI site at the 3′-endof the cassette coding sequence. The thermal amplification product wascloned into a pPCR-Script™ vector, and the sequence of the fusion wasverified by double-stranded sequencing. The SpeI/XhoI-fragmentcontaining the CryET33/CryET34 fusion peptide coding sequence was clonedinto an SpeI/XhoI-digested universal B.t. expression shuttle vectorpMON47407 indicated above, creating plasmid pMON38644 for expression ofthe CryET33/CryET34 fusion protein in B.t. strain EG10650, which is aB.t. strain which is deficient for the production of any insecticidalcrystal proteins. The ligation mixture from which pMON38644 was derivedwas transformed directly into the B.t. expression strain EG10650, andcolonies suspected of containing the expected plasmid were chosen forfurther analysis after selection on appropriate media.

[0214] One colony producing a protein of the expected size was selectedfor further analysis. Plasmid DNA from the transformant was isolated andcharacterized by restriction enzyme analysis. The EG10650 straincontaining the plasmid designated as pMON38644 (strain sIC2000) formedcrystal structures upon sporulation. Spores containing these crystalstructures were pelleted, washed and subjected to reducing SDS-PAGEanalysis, which revealed the presence of a protein of the expected size(43.8 kDa) which exhibited little if any signs of degradation. TheCryET33/CryET34 fusion protein crystals were submitted to qualitativebioassay against boll weevil upon solubilization into 10 mM NaHCO₃, pH10.0. Both soluble and insoluble fractions demonstrated bioactivityagainst boll weevil in a qualitative diet overlay bioassay.

Example 2 Construction of a CryET34/CryET33 Fusion in OrientationOpposite to the Native Operon with Insect Inhibitory Activity

[0215] This example illustrates the construction of a DNA sequenceencoding a CryET34 and CryET33 insect inhibitory fusion protein, andillustrates that the Colepteran inhibitory activity of a fusion proteinbetween CryET33 and CryET34 is independent of the orientation of the twoproteins within the fusion.

[0216] A CryET34/CryET33 fusion protein coding sequence was constructedby synthesizing a nucleic acid sequence having the CryET34 codingsequence located at the 5′-end, and the CryET33 sequence located at the3′-end. A BamHI/NheI linker coding for GSGGAS was also introducedbetween the two coding sequences. The sequence encoding theCryET34/CryET33 fusion protein was constructed as in example 1 above(see FIG. 2). The thermal amplification product sequence was cloned intoa pPCR-Script™ vector as in example 1, and the sequence was verified bydouble-stranded sequencing. The SpeI/XhoI-fragment containing theCryET34/CryET33 fusion peptide coding sequence was cloned into anSpeI/XhoI-digested universal B.t. expression vector pMON47407 resultingin the formation of plasmid pMON38646 which is useful for expression ofthe CryET34/CryET33 fusion protein in the B.t. crystal minus strainEG10650. The pMON38646 ligation mixture was transformed directly intoEG10650, and colonies suspected of containing the expected plasmid werechosen for further analysis after selection on the appropriate media.One colony containing a plasmid exhibiting the appropriatecharacteristics was designated as strain sIC2001.

[0217] Growth of strain sIC2001 containing pMON38646 (cryET34/cryET33fusion) revealed formation of crystal structures upon sporulation.Spores were pelleted, washed and subjected to reducing SDS-PAGEanalysis, which revealed the presence of a protein of the expected size(43.8 kDa).

Example 3 Development of ELISA Assay for CryET33/CryET34 Fusion Proteins

[0218] This example illustrates the development of an ELISA assay foruse in detecting and measuring the amount of a CryET33 and CryET34fusion protein in a sample.

[0219] An enzyme-linked immuno-sorbent assay was developed to evaluatethe expression of CryET33/CryET34 or CryET34/CryET33 fusion proteins ina sample or in an in planta sample. Polyclonal IgG, which had beenraised against a combination of both CryET33 and CryET34 proteins, waspurified from rabbit serum using Protein A affinity chromatography, andwas used as the capture or primary (1°) antibody (Ab). A secondary (2°)antibody capable of binding the 1° antibody was conjugated to analkaline phosphatase enzyme. A B.t.-expressed CryET33/CryET34 fusionprotein was used as standard reference material. A series of 96-wellimmunoassay plates were loaded using the CryET33/CryET34 fusion proteinstandard and different combinations of 1° and 2° Ab dilutions. A typicalCryET33/CryET34 standard curve is illustrated in FIG. 3. The appropriatedilutions were determined to be 1:500 for 1° Ab and 1:200 for 2° Ab. Theassay was tested qualitatively using tobacco plants expressingCryET33/CryET34 fusion protein and the results were confirmed by westernblot. These tobacco plants were then analyzed quantitatively and theresults were found to be reproducible upon repeating the assay (Table9). The assay has been used to evaluate expression in tobacco leaf,cotton callus, cotton leaf and cotton square. TABLE 9 Reproducibility ofthe CryET33/CryET34 fusion protein ELISA. Jan. 12, 2000, Jan. 19, 2000,Plant # Construct ppm ppm 1705-1 51713 0.39 0.39 1705-2 51713 0.23 0.181705-3 51713 0.23 0.23 1705-4 51713 0.15 0.15 1705-5 51713 0.46 0.421740-1 51719 1.27 1.32 1740-2 51719 1.03 1.02 1740-3 51719 3.15 3.211740-4 51719 1.15 1.16 1740-5 51719 0.96 0.96 1740-6 51719 1.29 1.351740-7 51719 1.82 1.86 1740-8 51719 2.73 2.63 1740-9 51719 1.64 1.60 1740-10 51719 1.75 1.82

Example 4 Expression and Bioactivity of CryET33/CryET34 Fusion Proteinin Cotton Callus Tissue

[0220] In order to quickly evaluate the in planta performance of theCryET33/CryET34 and CryET34/CryET33 fusion proteins, several constructswere made and expressed in cotton callus. In order to address possiblefolding or stability problems in plants, several parameters were varied.For example, two different linkers were incorporated between the BamHIand NheI restriction sites:

[0221] 1) (GGGS)₃ linker to allow for flexibility at the junction point;

[0222] 2) Lysine oxidase cleavage site linker which is known to becleaved in plants. This would allow the two proteins to fold correctlyin case the covalent linkage between the C-terminus of one protein andthe N-terminus of the other causes steric perturbance.

[0223] A chloroplast targeting sequence was also used, as well asvarious promoters. The constructs submitted for Agrobacterium-mediatedtransformation of cotton callus tissue are listed below in Table 10 (allconstructs contained an NPTII selectable marker). TABLE 10 PlantTransformation Plasmids Containing Various CryET33 and CryET34Translational Fusions Expression Cassette Description pMON # Promoter-ORF1- Linker- ORF2- terminator 51713 AtEF1a ET33 BamHI-NheI ET34 E951719 e35S ET33 BamHI-NheI ET34 E9 51739 e35S ET33 (GGGS)₃ ET34 E9 51740e35S ET33 LO ET34 E9 51758 AtEF1a ET34 BamHI-NheI ET33 E9

[0224] Transformed cotton callus tissue was lyophilized and subjected towestern blotting. Blots were probed with anti-CryET33/CryET34antibodies. The results demonstrate that CryET33/CryET34 fusionproteins, with either BamHI/NheI (pMON51713 and 51719),(GGGS)₃-(pMON51739) or lysine oxidase (pMON51740) linkers, are expressedin transformed cotton callus as judged by Western blot, and produce theprotein band of expected size (about 44 kDa). In this example, the bestexpressor was tissue transformed with plasmid pMON51719. Very littledegradation of the fusion protein to protrein fragments corresponding insize to the individual CryET33 (29 kDa) and CryET34 (14 kDa) proteinswas observed, indicating the stability of the fusions in cotton callustissue. A CryET34/CryET33 fusion, constructed in the double border planttransformation plasmid pMON51758, however, did not express any proteindetectable by Western blot in cotton callus tissue. The reason for thefailure of this construct to express the fusion protein in planta wasnot readily identifiable. It is believed however, because a CryET34/ET33fusion produced insecticidal protein of the expected size when expressedfrom a cassette introduced into EG10650, that successful expression ofCryET34/CryET33 fusion protein in cotton callus tissue could easily beachieved without undue experimentation.

[0225] The expression levels for CryET33/CryET34 fusion proteins inlyophilized cotton callus tissue as determined by ELISA are summarizedin Table 11. TABLE 11 Expression levels of CryET33/CryET34 fusions inlyophilized cotton callus. ET33/34 Date of fusion, pMON numbercollection Protein mg/g tissue 51713 Aug. 12, 1999 ET33/34 fusion 7.1751713 Sep. 14, 1999 ET33/34 fusion 7.66 51713 Oct. 12, 1999 ET33/34fusion 7.95 51713 Feb. 11, 2000 ET33/34 fusion 5.34 51713 Mar. 03, 2000ET33/34 fusion 5.02 51719 Jul. 22, 1999 ET33/34 fusion 14.01 51719 Sep.23, 1999 ET33/34 fusion 15.46 51719 Feb. 11, 2000 ET33/34 fusion 13.1451719 Mar. 03, 2000 ET33/34 fusion 14.53 51739 Nov. 16, 1999 ET33/34fusion 18.68 51739 Jan. 12, 2000 ET33/34 fusion 14.40 51739 Feb. 11,2000 ET33/34 fusion 6.15 51739 Mar. 03, 2000 ET33/34 fusion 5.62 51740Nov. 16, 1999 ET33/34 fusion 7.31 51740 Jan. 12, 2000 ET33/34 fusion6.51 51740 Feb. 11, 2000 ET33/34 fusion 2.64 51740 Mar. 03, 2000 ET33/34fusion 2.38 51758 Feb. 11, 2000 ET34/33 fusion 0.00 51758 Mar. 03, 2000ET34/33 fusion 0.00

[0226] As indicated from the data in Table 11, the highest expression ofa CryET33/CryET34 fusion protein was consistently achieved when usingpMON51719. This result is consistent with western blotting data.

[0227] In order to determine the bioactivity of the lyophilized callustissues, the transformed callus tissues were tested in a boll weevildiet-overlay bioassay. The results of three independent bioassays, andthe expression levels for the lyophilized cotton callus tissuesexpressing CryET33/CryET34 fusion protein, are shown in Tables 12-14. Asindicated from the data in Tables 12-14, callus tissue transformed withplasmid pMON51739 or plasmid pMON51719 consistently demonstratedsignificant boll weevil activity. In addition, the results shown inTables 12-14 demonstrate that the transformed tissues exhibiting thegreatest boll weevil activity correlated well with elevated expressionlevels as measured by ELISA, so that expression levels of the fusionproteins could be used to screen for transformation events exhibitingcommercial levels of fusion protein expression and coleopteran insectinhibitory bioactivity. TABLE 12 Boll Weevil Bioactivity of LyophilizedCotton Callus Tissues Transformed to Express CryET33/CryET34FusionProtein pMON-date of collection % Mortality ELISA, ppm 39778* 0.00 051713-Aug. 12, 1999 0.00 7.17 51713-Sep. 14, 1999 6.25 7.66 51713-Oct.12, 1999 6.67 7.95 51719-Jan. 12, 2000 16.67 14.01 51739-Nov. 16, 199935.29 18.68 51739-Jan. 12, 2000 25.00 14.4 51740-Nov. 16, 1999 5.88 7.3151740-Jan. 12, 2000 6.25 6.51

[0228] TABLE 13 Boll Weevil Bioactivity of Lyophilized Callus TissuesTransformed to Express CryET33/CryET34 pMON-date of collection %Mortality ELISA, ppm 39778* 0.00 0.00 51713-Feb. 11, 2000 0.00 5.3451713-Mar. 23, 2000 0.00 5.02 51719-Feb. 11, 2000 31.25 13.14 51719-Mar.23, 2000 40.00 14.53 51739-Feb. 11, 2000 6.67 6.15 51739-Mar. 23, 20000.00 5.62 51740-Feb. 11, 2000 0.00 2.64 51740-Mar. 23, 2000 6.25 2.3851758-Feb. 11, 2000 0.00 0.00 51758-Mar. 23, 2000 6.67 0.00

[0229] TABLE 14 Boll Weevil Bioactivity of Lyophilized Callus TissuesTransformed to Express CryET33/CryET34 pMON-date of collection %Mortality ELISA, ppm 39778* 0.00 0 51713-Aug. 12, 1999 6.67 5.9151713-Sep. 14, 1999 7.14 7.01 51713-Oct. 12, 1999 0.00 7.26 51713-Mar.3, 2000 0.00 5.02 51719-Jul. 22, 1999 25.00 10.96 51719-Sep. 23 199926.67 11.72 51719-Mar. 3, 2000 31.25 14.53 51739-Nov. 16, 1999 28.5719.47 51739-Feb. 11, 2000 0.00 6.15 51739-Mar. 3, 2000 0.00 5.6251740-Nov. 16, 1999 14.29 9.93 51740-Jan. 12, 2000 6.25 4.84 51740-Mar.3, 2000 0.00 2.38 51758-Mar. 3, 2000 0.00 0

Example 5 Bioactivity of CryET33/CryET34 Fusion Protein Expressed inCotton Plants

[0230] In order to evaluate expression and bioactivity ofCryET33/CryET34 fusion protein in a target plant, pMON51713 andpMON51719 were submitted for cotton transformation and plantregeneration (all constructs contained a NPTII selectable marker).

[0231] The expression levels were determined for R₀ plants by ELISA infresh cotton leaf tissue, and then in fresh cotton squares. Severalplants were determined to express levels of CryET33/CryET34 fusionprotein above LC₅₀ values for CryET33/CryET34 fusion protein (1-5 ppm).These results are presented in Table 15. TABLE 15 Expression ofCryET33/CryET34 fusion protein in fresh cotton tissue* Plant ELISA valuein leaf ELISA value in square (pMON-plant number) tissue, ppm tissue,ppm 51713-S011036 3.40 2.27 51719-S011132 8.22 19.59 51719-S011154 5.521.39 51719-S011207 4.93 1.53 51719-S011339 8.94 ND 51719-S011470 7.90 ND51719-S011482 6.43 ND 51719-S011480 6.13 ND 51719-S011481 5.44 ND51719-S011664 8.22 ND 51719-S011875 13.97 ND 51719-S012091 6.28 ND51719-S012253 6.53 ND

[0232] Bioactivity of cotton squares expressing CryET33/CryET34 fusionprotein against boll weevil for several available plants was testedusing lyophilized tissue in diet-overlay bioassay (3% callus tissue inAgar). The results for plant S011132 are presented in Table 16, whichdemonstrates that plant S011132 (pMON51719, ET33/ET34 fusion withBamHI/NheI linker driven by e35S promoter) exhibits commercial levels ofactivity against boll weevil. The results further suggest theCryET33/CryET34 fusion proteins can be highly efficacious in cottonsquares which are the primary targets of boll weevil infestation. TABLE16 CryET33/CryET34 Fusion Protein Bioactivity Against Boll Weevil SampleMortality, % Stunting, % C312 7.7 0 51719-S011132 60 80.7 ET33/ET34 PPM25 78.9

[0233] Eleven six-week-old R1 plants selected after Agrobacteriummediated transformation with the plasmid pMON51719, i.e., containing aninsecticidal fusion of CryET33 and CryET34 linked in frame by a GSGGASlinker, were transferred to a growth chamber in which temperature andhumidity conditions were precisely controlled. The plants exhibited arandom range of expression levels. Two plants were observed to expressno detectable insecticidal protein, and one plant was a expressed verylow levels of the fusion protein. Four plants Coker C312 non-transgenicplants were used as negative controls. All plants were infested withadults boll weevils on a weekly basis for four weeks. Flaring squaresfrom each plant were collected in individual plastic containers, anddissected after a period of three weeks in order to enumerate the numberof larval and adult weevils. In all, each plant was sampled individuallyfive times. Leaf and square tissue samples were obtained at the outsetof the experiment and fusion protein levels were determined by ELISA.

[0234] The results demonstrated in vivo activity of an ET33/ET34 fusionprotein containing a GSGGAS linker against cotton boll weevils. TheELISA data collected from protein fractions from leaf and square tissuesamples from each plant tested correlated well with the observedbioactivity of the plants exhibiting the highest ELISA values. Bollretention also correlated well with the observed expression profiles, inthat the plants exhibiting the greatest level of fusion proteinexpression as judged by ELISA were the plants least susceptible to bolldrop upon weevil infestation. In this example, in order to mimic orexceed a field level high pressure infestation, the plants weresubjected to four independent infestations of adult weevils. Thisartificial infestation level was much greater than the infestation thatwould typically be observed under wild infestation conditions.

[0235] One undesireable consequence of the expression of this particularET33/34 fusion in this plant line was an aberrant plant phenotype. Thecotton plants expressing the greatest levels of the ET33/ET34 fusionexhibited an obvious uncharacteristic phenotype. Plants exhibiting alower level of expression had less severe symptoms, however, all plantsderived from this transformation event exhibited some level of theobserved symptoms. The principal morphological change observed in theseplants was a swelling of the stems. In the most extreme cases there wasa shorting of the internode distance resulting in slightly shorterstature. There did not seem to be any major impact on plant fertility.The observed phenotype could be specific to this particulartransformation event and is likely attributable to the site of insertionof the cassette expressing the transgene.

Example 6 Fusion of tIC100/tIC101 Insect Inhibitory Proteins

[0236] The binary insecticidal toxin identified herein and designated asopen reading frames producing the proteins tIC100 and tIC101, is derivedfrom Bacillus thuringiensis strain EG9328. The native Bacillusthuringiensis DNA sequence contained a frame-shift in the codingsequence for the tIC100 protein. This frame-shift was altered bysite-directed mutagenesis to produce the coding sequence as set forth inSEQ ID NO:1, which resulted in the generation of an operon which, whenexpressed in Bacillus thuringiensis strain EG10650 from plasmid pIC10000(strain sIC1000), encodes a Coleopteran-inhibitory product comprisingtwo proteins—tIC100 (29 kDa) and tIC101 (14 kDa).

[0237] Therefore, tIC100 is a protein derived from a cryptic B.thuringiensis DNA sequence. The cryptic tIC100 coding sequence is a partof an operon containing the tIC101 coding sequence, and is adjacent toand upstream of the coding sequence for tIC101. The cryptic sequenceupstream of tIC101 contains the complete coding sequence for tIC100except that a single guanosine residue at position 84 of the nativecryptic tIC100 coding sequence as set forth in SEQ ID NO:27 causes thetIC100 coding sequence to be out of frame. The frameshift was eliminatedby removing the single guanosine residue at position 84 to create thenovel tIC100 coding sequence as set forth in SEQ ID NO:1. Overlappingthermal amplification mutagenesis was employed to repair the tIC100reading frame. Four oligonucleotides were synthesized to complete thereconstruction of a functional coding sequence for tIC100. Two reversecomplementary primers, SEQ ID NO:28 and SEQ ID NO:29, were synthesizedwhich spanned the target site sequence, i.e., the guanosine residue tobe removed from the cryptic B.t. sequence. Two additional primers weresynthesized to take advantage of sequences downstream within the cryptictIC100 coding sequence and upstream of the proposed promoter sequencefor the operon. SEQ ID NO:30 is complementary to nucleotide positions625-639 in tIC100 as shown in SEQ ID NO:1, and was used with SEQ IDNO:28 in a thermal amplification reaction with the cryptic tIC100 as atemplate to produce a first product which contains the correctedsequence from just upstream of the frameshift correction point or targetsite sequence to just downstream of a unique PstI site in the tIC100coding sequence, located at nucleotide positions 247-252 of SEQ ID NO:1.The other oligonucleotide primer, SEQ ID NO:29, was used along with SEQID NO:31 in a thermal amplification reaction using the cryptic tIC100sequence as a template to produce a second product which also containsthe corrected sequence at one end and an EcoRI restriction site at thedistal end of the product. The two amplification products were thenmixed into a third thermal amplification reaction along with primerscorresponding to SEQ ID NO:30 and SEQ ID NO:31, denatured and thenallowed to anneal, a portion of the annealed products representing onestrand of the first product annealed at one end to the complementary endof one strand of the other amplification product. The overlap/annealedsequence from both products represents the reverse complementarysequences of SEQ ID NO:28 and SEQ ID NO:29. Elongation of thosesequences in the thermal amplification reaction produced a sequencewhich was then amplified by the oligos represented by SEQ ID NO:30 andSEQ ID NO:31 to produce a third product, which was purified, digestedwith PstI and EcoRI and inserted into the native cryptic sequence inplace of the native frame-shifted sequence to produce the novelfunctional sequence encoding the tIC100 and tIC101 coleopteraninhibitory binary toxin peptides.

[0238] The amino acid sequence of the tIC100 and tIC101 binary peptidetoxin is similar to the amino acid sequence of the CryET33 and CryET34binary peptide toxin. CryET33 is a comparative counterpart to CrytIC100,and CryET34 is a comparative counterpart to CrytIC101. The amino acidsequence of tIC100 was 74% identical to the amino acid sequence ofCryET33, and the amino acid sequence of tIC101 was about 82.5% identicalto the amino acid sequence of CryET34. It was postulated that tIC100 andtIC101 may share common structural and functional properties withCryET33 and CryET34 because of the similarity between the amino acidsequences of these proteins and that these proteins would have similarbioactivity. In fact, insect inhibitory assays using tIC100 and tIC101herein and completed as described in Examples 9 and 10 of U.S. Pat. No.6,063,756 demonstrated insect inhibitory activity.

[0239] In view of the insect inhibitory activity exhibited by the binarytoxin protein CrytIC100 and CrytIC101, and the similarities between theCryET33/CryET34 binary toxin protein, it was further postulated that afusion protein could be constructed in a manner similar to thosedescribed in Examples 1 and 2 above. Several fusions were designed andconstructed. Two of these fusions were designed similarly to theCryET33/CryET34 fusions. That is, the tIC100 and tIC101 proteins werefused in both orientations (i.e., tIC100-tIC101 and tIC101-tIC100) andseparated by a short hydrophilic linker (Gly-Ser-Gly-Gly-Ala-Ser). Thenucleic acid sequence encoding the linker is embraced by unique BamHIand NheI endonuclease restriction sites. Two other fusions were designedwith a short Gly-Gly linker since this configuration more closelyresembles the distance between the tIC100 and tIC101 sequences in thenative B.t. operon.

[0240] These nucleotide sequences encoding the tIC100/tIC101 fusionswere made by overlapping thermal amplification mutagenesis, cloned intothe B.t. expression vector pMON47407 and expressed in B.t. strainEG10650. Strain numbers have been assigned to these expression strainsas indicated in Table 17. TABLE 17 B.T. STRAINS CONTAINING PLASMIDSENCODING ET33/ET34 AND TIC100/TIC101 FUSIONS Strain number pMON #Description of Fusion Expression Cassette sIC2000 38644 ET33-GSGGAS-ET34sIC2001 38646 ET34-GSGGAS-ET33 sIC2002 38651ET33-GSPALLKEAPRAEEELPPAS-ET34 sIC2003 38652 ET33-(GGGS)₃-ET34 sIC200638653 tIC100-GSGGAS-tIC101 sIC2007 38654 tIC100-GG-tIC101 sIC2008 38655tIC101-GG-tIC100 sIC2010 38657 tIC101-GSGGAS-tIC100

[0241] The tIC100/tIC101 fusions were expressed and identified withinthe spores-crystal fraction of sporulated B.t. expression strains.SDS-PAGE analysis revealed the presence of the band of expected size (44kDa), which is not present in the host strain (EG10650) alone.

[0242] The spores-crystal fraction suspensions of tIC100/tIC101 fusionswere quantitated using spot densitometry and submitted for adiet-overlay bioassay against boll weevil in parallel withCryET33/CryET34 fusions. These results are shown in FIG. 3. FIG. 3demonstrates that the tIC100/tIC101 fusions (sIC2006, sIC2007 andsIC2008) are approximately as active as the CryET33/CryET34 fusions(sIC2000 and sIC2001).

[0243] All of the compositions- and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions, methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims. Accordingly, the exclusiverights sought to be patented are as described in the claims below.

REFERENCES CITED

[0244] U.S. Pat. No. 4,766,203 August, 1988 Krieg et al. 530/370.

[0245] U.S. Pat. No. 4,771,131 September, 1988 Hernstadt et al. 536/27.

[0246] U.S. Pat. No. 4,797,279 January, 1989 Karamata et al. 424/93.

[0247] U.S. Pat. No. 4,910,016 March, 1990 Gaertner et al. 424/93.

[0248] U.S. Pat. No. 4,966,765 October, 1990 Payne et al. 424/93.

[0249] U.S. Pat. No. 4,996,155 February, 1991 Sick et al. 435/252.

[0250] U.S. Pat. No. 4,999,192 March, 1991 Payne et al. 424/93.

[0251] U.S. Pat. No. 5,006,336 April, 1991 Payne 424/93.

[0252] U.S. Pat. No. 5,024,837 June, 1991 Donovan et al. 424/93.

[0253] U.S. Pat. No. 5,071,654 December, 1991 English 424/405.

[0254] U.S. Pat. No. 5,143,905 September, 1992 Sivasubramanian et al.514/21.

[0255] U.S. Pat. No. 5,173,409 December, 1992 Englsih 435/71.

[0256] U.S. Pat. No. 5,187,091 February, 1993 Donovan et al. 435/240.

[0257] U.S. Pat. No. 5,264,364 November, 1993 Donovan et al. 435/252.

[0258] U.S. Pat. No. 5,286,486 February, 1994 Payne et al. 424/93.

[0259] U.S. Pat. No. 5,338,544 August, 1994 Donovan 424/93.

[0260] U.S. Pat. No. 5,356,623 October, 1994 von Tersch et al. 424/93.

[0261] U.S. Pat. No. 5,378,625 January, 1995 Donovan et al. 435/252.

[0262] U.S. Pat. No. 5,382,429 January, 1995 Donovan et al. 424/94.

[0263] U.S. Pat. No. 5,384,253 January, 1995 Krzyzek et al. 435/172.

[0264] U.S. Pat. No. 5,441,884 August, 1995 Baum 435/252.

[0265] U.S. Pat. No. 6,063,756 May, 2000 Donovan et al. 514/2.

[0266] U.S. Pat. No. 6,083,499 July, 2000 Narva et al. 424/93.

[0267] U.S. Pat. No. 6,037,522 March, 2000 Jinjiang et al. 800/287.

Foreign Patent Documents

[0268] 0318143 A2 October, 1988 EP.

[0269] 0324254 A1 December, 1988 EP.

[0270] 0382990 A1 February, 1989 EP.

[0271] WO89/07605 August, 1989 WO.

[0272] WO90/13651 November, 1990 WO.

[0273] WO91/07481 May, 1991 WO.

[0274] WO91/14778 October, 1991 WO.

[0275] WO92/13954 August, 1992 WO.

[0276] WO94/13785 June, 1994 WO.

[0277] WO94/16079 July, 1994 WO.

[0278] WO95/02693 January, 1995 WO.

[0279] WO95/06730 March, 1995 WO.

[0280] WO95/30752 November, 1995 WO.

[0281] WO95/30753 November, 1995 WO.

[0282] WO95/35378 December, 1995 WO.

[0283] WO00/066,742 November, 2000 WO.

[0284] WO01/14417(A2) March, 2001 WO.

Other References

[0285] Cidaria et al., “A novel strain of Bacillus thuringiensis (NCIMB40152) active against coleopteran insects,” FEMS Microbiology Letters,81:129-134, 1991.

[0286] Cody et al., “Purification and Crystallization of Insecticidalδ-Endotoxin CyrIIIB2 From Bacillus thuringiensis,” Proteins: Structure,Function and Genetics, 14:324, 1992.

[0287] Donovan et al., “Characterization of two genes encoding Bacillusthuringiensis insecticidal crystal proteins toxic to coleopteraspecies,” Applied and Environmental Microbiology, 58(12):3921-3927,1992.

[0288] Donovan et al., “Isolation and characterizations of EG2158, a newstrain of Bacillus thuringiensis toxic to coleopteran larvae, andnucleotide sequence of the toxin gene,” Mol Gen Genet, 214:365-372,1988.

[0289] Lambert et al., “A Bacillus thuringiensis insecticidal crystalprotein with a high activity against members of the family noctuidae,”Applied and Environmental Microbiology, 62(1):80-86, 1996.

[0290] Lambert et al., “Novel Bacillus thuringiensis insecticidalcrystal protein with a silent activity against coleopteran larvae,”Applied and Environmental Microbiology, 58(8):2536-2542, 1992.

[0291] Lambert et al., “Nucleotide sequence of gene cryIIID encoding anovel coleopteran-active crystal protein from strain BTI109P of Bacillusthuringiensis subsp. kurstaki,” Gene, 110:131-132, 1992.

[0292] Sick et al., “Nucleotide sequence of a coleopteran-active toxingene from a new isolate of Bacillus thuringiensis subsp. tolworthi,”Nucleic Acids Research, 18(5):1305, 1989.

[0293] Von Tersch et al., “Membrane-Permeabilizing Activies of Bacillusthuringiensis Coleopteran-Active Toxin CryIIIB2 and CryIIIB2 Domain IPeptide,” Applied and Environmental Microbiology, 60(10):3711-3717,1994.

[0294] Cooper, Biotechnology and the Law. Deerfield: CBC. vol. 1, pp.5B-41 through 5B-43, 1992.

[0295] Ely, “The engineering of plants to express Bacillus thuringiensisδ-endotoxins,” In: Bacillus thuringiensis, An EnvironmentalBiopesticide: Theory and Practice, Entwistle, et al., Eds., Chichester,Wiley & Sons, pp. 105-124, 1993.

[0296] International Search Report dated Feb. 20, 1998 (PCT/US97/17600)(MECO:203P).

[0297] Johnson et al., “Insecticidal activity of EG-4961, a novel strainof Bacillus thuringiensis toxic to larvae and adults of Southern CornRootworm (Coleoptera: Chrysomelidae) and Colorado Potato Beetle(Coleoptera: Chrysomelidae),” J. Economic Entomol., 86(2):330-333, 1993.

[0298]

1 33 1 804 DNA Bacillus thuringiensis CDS (1)..(804) tIC100 codingsequence 1 atg gga att atc aac att caa gac gaa att aat gac tac atg aaaggt 48 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp Tyr Met Lys Gly 15 10 15 atg tat ggt gca aca tct gtt aaa agc act tat gac ccc tca ttc aaa96 Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 20 2530 gta ttt aac gaa tct gtg aca cct caa tat gat gtg att cca aca gaa 144Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp Val Ile Pro Thr Glu 35 40 45cct gta aat aat cat att act act aaa gta ata gat aat cca ggg act 192 ProVal Asn Asn His Ile Thr Thr Lys Val Ile Asp Asn Pro Gly Thr 50 55 60 tcagaa gta acc agt aca gta acg ttc aca tgg acg gaa acc gac act 240 Ser GluVal Thr Ser Thr Val Thr Phe Thr Trp Thr Glu Thr Asp Thr 65 70 75 80 gtaacc tct gca gtg act aaa ggg tat aaa gtc ggt ggt tca gta agc 288 Val ThrSer Ala Val Thr Lys Gly Tyr Lys Val Gly Gly Ser Val Ser 85 90 95 tca aaagca act ttt aaa ttt gct ttt gtt act tct gat gtt act gta 336 Ser Lys AlaThr Phe Lys Phe Ala Phe Val Thr Ser Asp Val Thr Val 100 105 110 act gtatca gca gaa tat aat tat agt aca aca gaa aca aca aca aaa 384 Thr Val SerAla Glu Tyr Asn Tyr Ser Thr Thr Glu Thr Thr Thr Lys 115 120 125 aca gataca cgc aca tgg acg gat tcg acg aca gta aaa gcc cct cca 432 Thr Asp ThrArg Thr Trp Thr Asp Ser Thr Thr Val Lys Ala Pro Pro 130 135 140 aga actaat gta gaa gtt gca tat att atc caa act gga aat tat aac 480 Arg Thr AsnVal Glu Val Ala Tyr Ile Ile Gln Thr Gly Asn Tyr Asn 145 150 155 160 gttccg gtt aat gta gag tct gat atg act gga acg cta ttt tgc aga 528 Val ProVal Asn Val Glu Ser Asp Met Thr Gly Thr Leu Phe Cys Arg 165 170 175 gggtat aga gat ggt gca cta att gca gcg gct tat gtt tct ata aca 576 Gly TyrArg Asp Gly Ala Leu Ile Ala Ala Ala Tyr Val Ser Ile Thr 180 185 190 gattta gca gat tac aat cct aat ttg ggt ctt aca aat gaa ggg aat 624 Asp LeuAla Asp Tyr Asn Pro Asn Leu Gly Leu Thr Asn Glu Gly Asn 195 200 205 ggggtt gct cat ttt aaa ggt gaa ggt tat ata gag ggt gcg caa ggc 672 Gly ValAla His Phe Lys Gly Glu Gly Tyr Ile Glu Gly Ala Gln Gly 210 215 220 ttaaga agc tac att caa gtt aca gaa tat cca gtg gat gat aat ggc 720 Leu ArgSer Tyr Ile Gln Val Thr Glu Tyr Pro Val Asp Asp Asn Gly 225 230 235 240aga cat tcg ata cca aaa act tat ata att aaa ggt tca tta gca ccc 768 ArgHis Ser Ile Pro Lys Thr Tyr Ile Ile Lys Gly Ser Leu Ala Pro 245 250 255aat gtt act tta ata aat gat aga aag gaa ggt aga 804 Asn Val Thr Leu IleAsn Asp Arg Lys Glu Gly Arg 260 265 2 268 PRT Bacillus thuringiensis 2Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp Tyr Met Lys Gly 1 5 1015 Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 20 2530 Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp Val Ile Pro Thr Glu 35 4045 Pro Val Asn Asn His Ile Thr Thr Lys Val Ile Asp Asn Pro Gly Thr 50 5560 Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr Glu Thr Asp Thr 65 7075 80 Val Thr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly Gly Ser Val Ser 8590 95 Ser Lys Ala Thr Phe Lys Phe Ala Phe Val Thr Ser Asp Val Thr Val100 105 110 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Glu Thr Thr ThrLys 115 120 125 Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val Lys AlaPro Pro 130 135 140 Arg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr GlyAsn Tyr Asn 145 150 155 160 Val Pro Val Asn Val Glu Ser Asp Met Thr GlyThr Leu Phe Cys Arg 165 170 175 Gly Tyr Arg Asp Gly Ala Leu Ile Ala AlaAla Tyr Val Ser Ile Thr 180 185 190 Asp Leu Ala Asp Tyr Asn Pro Asn LeuGly Leu Thr Asn Glu Gly Asn 195 200 205 Gly Val Ala His Phe Lys Gly GluGly Tyr Ile Glu Gly Ala Gln Gly 210 215 220 Leu Arg Ser Tyr Ile Gln ValThr Glu Tyr Pro Val Asp Asp Asn Gly 225 230 235 240 Arg His Ser Ile ProLys Thr Tyr Ile Ile Lys Gly Ser Leu Ala Pro 245 250 255 Asn Val Thr LeuIle Asn Asp Arg Lys Glu Gly Arg 260 265 3 378 DNA Bacillus thuringiensisCDS (1)..(378) tIC101 3 atg aca gta tat aac gta act ttt acc att aaa ttctat aat gaa ggt 48 Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe TyrAsn Glu Gly 1 5 10 15 gaa tgg ggg ggg cca gaa cct tac ggt aag ata tatgca tac ctt caa 96 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr AlaTyr Leu Gln 20 25 30 aat cca gat cat aat ttc gaa att tgg tca caa gat aattgg ggg aag 144 Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn TrpGly Lys 35 40 45 gat acg cct gag aaa agt tct cac act caa aca att aaa ataagt agc 192 Asp Thr Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys Ile SerSer 50 55 60 cca aca ggg ggg cct ata aac caa atg tgt ttt tat ggt gat gtaaaa 240 Pro Thr Gly Gly Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys65 70 75 80 gaa tac gac gta gga aat gca gat gat gtt ctc gcc tat cca agtcaa 288 Glu Tyr Asp Val Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro Ser Gln85 90 95 aaa gta tgc agt acg cct ggc aca aca ata agg ctt aac gga gat gag336 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu 100105 110 aaa ggt tct tat ata cag att aga tat tcc ttg gcc cca gct 378 LysGly Ser Tyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala 115 120 125 4 126PRT Bacillus thuringiensis 4 Met Thr Val Tyr Asn Val Thr Phe Thr Ile LysPhe Tyr Asn Glu Gly 1 5 10 15 Glu Trp Gly Gly Pro Glu Pro Tyr Gly LysIle Tyr Ala Tyr Leu Gln 20 25 30 Asn Pro Asp His Asn Phe Glu Ile Trp SerGln Asp Asn Trp Gly Lys 35 40 45 Asp Thr Pro Glu Lys Ser Ser His Thr GlnThr Ile Lys Ile Ser Ser 50 55 60 Pro Thr Gly Gly Pro Ile Asn Gln Met CysPhe Tyr Gly Asp Val Lys 65 70 75 80 Glu Tyr Asp Val Gly Asn Ala Asp AspVal Leu Ala Tyr Pro Ser Gln 85 90 95 Lys Val Cys Ser Thr Pro Gly Thr ThrIle Arg Leu Asn Gly Asp Glu 100 105 110 Lys Gly Ser Tyr Ile Gln Ile ArgTyr Ser Leu Ala Pro Ala 115 120 125 5 1188 DNA Artificial sequenceRecombinant fusion protein 5 atg aca gta tat aac gta act ttt acc att aaattc tat aat gaa ggt 48 Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys PheTyr Asn Glu Gly 1 5 10 15 gaa tgg ggg ggg cca gaa cct tac ggt aag atatat gca tat ctt caa 96 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile TyrAla Tyr Leu Gln 20 25 30 aat cca gat cat aat ttc gaa att tgg tca caa gataat tgg ggg aag 144 Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp AsnTrp Gly Lys 35 40 45 gat acg cct gag aaa agt tct cac act caa aca att aaaata agt agc 192 Asp Thr Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys IleSer Ser 50 55 60 cca aca ggg ggg cct ata aac caa atg tgt ttt tat ggt gatgta aaa 240 Pro Thr Gly Gly Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp ValLys 65 70 75 80 gaa tac gac gta gga aat gca gat gat gtt ctc gcc tat ccaagt caa 288 Glu Tyr Asp Val Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro SerGln 85 90 95 aaa gta tgc agt acg cct ggc aca aca ata agg ctt aac gga gatgag 336 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu100 105 110 aaa ggt tct tat ata cag att aga tat tcc ttg gcc cca gct ggtgga 384 Lys Gly Ser Tyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala Gly Gly115 120 125 atg gga att atc aac att caa gac gaa att aat gac tac atg aaaggt 432 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp Tyr Met Lys Gly130 135 140 atg tat ggt gca aca tct gtt aaa agc act tat gac ccc tca ttcaaa 480 Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys145 150 155 160 gta ttt aac gaa tct gtg aca cct caa tat gat gtg att ccaaca gaa 528 Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp Val Ile Pro ThrGlu 165 170 175 cct gta aat aat cat att act act aaa gta ata gat aat ccaggg act 576 Pro Val Asn Asn His Ile Thr Thr Lys Val Ile Asp Asn Pro GlyThr 180 185 190 tca gaa gta acc agt aca gta acg ttc aca tgg acg gaa accgac act 624 Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr Glu Thr AspThr 195 200 205 gta acc tct gca gtg act aaa ggg tat aaa gtc ggt ggt tcagta agc 672 Val Thr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly Gly Ser ValSer 210 215 220 tca aaa gca act ttt aaa ttt gct ttt gtt act tct gat gttact gta 720 Ser Lys Ala Thr Phe Lys Phe Ala Phe Val Thr Ser Asp Val ThrVal 225 230 235 240 act gta tca gca gaa tat aat tat agt aca aca gaa acaaca aca aaa 768 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Glu Thr ThrThr Lys 245 250 255 aca gat aca cgc aca tgg acg gat tcg acg aca gta aaagcc cct cca 816 Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val Lys AlaPro Pro 260 265 270 aga act aat gta gaa gtt gca tat att atc caa act ggaaat tat aac 864 Arg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr Gly AsnTyr Asn 275 280 285 gtt ccg gtt aat gta gag tct gat atg act gga acg ctattt tgc aga 912 Val Pro Val Asn Val Glu Ser Asp Met Thr Gly Thr Leu PheCys Arg 290 295 300 ggg tat aga gat ggt gca cta att gca gcg gct tat gtttct ata aca 960 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Ala Tyr Val SerIle Thr 305 310 315 320 gat tta gca gat tac aat cct aat ttg ggt ctt acaaat gaa ggg aat 1008 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Gly Leu Thr AsnGlu Gly Asn 325 330 335 ggg gtt gct cat ttt aaa ggt gaa ggt tat ata gagggt gcg caa ggc 1056 Gly Val Ala His Phe Lys Gly Glu Gly Tyr Ile Glu GlyAla Gln Gly 340 345 350 tta aga agc tac att caa gtt aca gaa tat cca gtggat gat aat ggc 1104 Leu Arg Ser Tyr Ile Gln Val Thr Glu Tyr Pro Val AspAsp Asn Gly 355 360 365 aga cat tcg ata cca aaa act tat ata att aaa ggttca tta gca ccc 1152 Arg His Ser Ile Pro Lys Thr Tyr Ile Ile Lys Gly SerLeu Ala Pro 370 375 380 aat gtt act tta ata aat gat aga aag gaa ggt aga1188 Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly Arg 385 390 395 6 396PRT Artificial sequence Recombinant fusion protein 6 Met Thr Val Tyr AsnVal Thr Phe Thr Ile Lys Phe Tyr Asn Glu Gly 1 5 10 15 Glu Trp Gly GlyPro Glu Pro Tyr Gly Lys Ile Tyr Ala Tyr Leu Gln 20 25 30 Asn Pro Asp HisAsn Phe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys 35 40 45 Asp Thr Pro GluLys Ser Ser His Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 Pro Thr Gly GlyPro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 Glu Tyr AspVal Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro Ser Gln 85 90 95 Lys Val CysSer Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu 100 105 110 Lys GlySer Tyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala Gly Gly 115 120 125 MetGly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp Tyr Met Lys Gly 130 135 140Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 145 150155 160 Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp Val Ile Pro Thr Glu165 170 175 Pro Val Asn Asn His Ile Thr Thr Lys Val Ile Asp Asn Pro GlyThr 180 185 190 Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr Glu ThrAsp Thr 195 200 205 Val Thr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly GlySer Val Ser 210 215 220 Ser Lys Ala Thr Phe Lys Phe Ala Phe Val Thr SerAsp Val Thr Val 225 230 235 240 Thr Val Ser Ala Glu Tyr Asn Tyr Ser ThrThr Glu Thr Thr Thr Lys 245 250 255 Thr Asp Thr Arg Thr Trp Thr Asp SerThr Thr Val Lys Ala Pro Pro 260 265 270 Arg Thr Asn Val Glu Val Ala TyrIle Ile Gln Thr Gly Asn Tyr Asn 275 280 285 Val Pro Val Asn Val Glu SerAsp Met Thr Gly Thr Leu Phe Cys Arg 290 295 300 Gly Tyr Arg Asp Gly AlaLeu Ile Ala Ala Ala Tyr Val Ser Ile Thr 305 310 315 320 Asp Leu Ala AspTyr Asn Pro Asn Leu Gly Leu Thr Asn Glu Gly Asn 325 330 335 Gly Val AlaHis Phe Lys Gly Glu Gly Tyr Ile Glu Gly Ala Gln Gly 340 345 350 Leu ArgSer Tyr Ile Gln Val Thr Glu Tyr Pro Val Asp Asp Asn Gly 355 360 365 ArgHis Ser Ile Pro Lys Thr Tyr Ile Ile Lys Gly Ser Leu Ala Pro 370 375 380Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly Arg 385 390 395 7 1200 DNAArtificial sequence Recombinant fusion protein 7 atg gga att atc aac attcaa gac gaa att aat gac tac atg aaa ggt 48 Met Gly Ile Ile Asn Ile GlnAsp Glu Ile Asn Asp Tyr Met Lys Gly 1 5 10 15 atg tat ggt gca aca tctgtt aaa agc act tat gac ccc tca ttc aaa 96 Met Tyr Gly Ala Thr Ser ValLys Ser Thr Tyr Asp Pro Ser Phe Lys 20 25 30 gta ttt aac gaa tct gtg acacct caa tat gat gtg att cca aca gaa 144 Val Phe Asn Glu Ser Val Thr ProGln Tyr Asp Val Ile Pro Thr Glu 35 40 45 cct gta aat aat cat att act actaaa gta ata gat aat cca ggg act 192 Pro Val Asn Asn His Ile Thr Thr LysVal Ile Asp Asn Pro Gly Thr 50 55 60 tca gaa gta acc agt aca gta acg ttcaca tgg acg gaa acc gac act 240 Ser Glu Val Thr Ser Thr Val Thr Phe ThrTrp Thr Glu Thr Asp Thr 65 70 75 80 gta acc tct gca gtg act aaa ggg tataaa gtc ggt ggt tca gta agc 288 Val Thr Ser Ala Val Thr Lys Gly Tyr LysVal Gly Gly Ser Val Ser 85 90 95 tca aaa gca act ttt aaa ttt gct ttt gttact tct gat gtt act gta 336 Ser Lys Ala Thr Phe Lys Phe Ala Phe Val ThrSer Asp Val Thr Val 100 105 110 act gta tca gca gaa tat aat tat agt acaaca gaa aca aca aca aaa 384 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr ThrGlu Thr Thr Thr Lys 115 120 125 aca gat aca cgc aca tgg acg gat tcg acgaca gta aaa gcc cct cca 432 Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr ThrVal Lys Ala Pro Pro 130 135 140 aga act aat gta gaa gtt gca tat att atccaa act gga aat tat aac 480 Arg Thr Asn Val Glu Val Ala Tyr Ile Ile GlnThr Gly Asn Tyr Asn 145 150 155 160 gtt ccg gtt aat gta gag tct gat atgact gga acg cta ttt tgc aga 528 Val Pro Val Asn Val Glu Ser Asp Met ThrGly Thr Leu Phe Cys Arg 165 170 175 ggg tat aga gat ggt gca cta att gcagcg gct tat gtt tct ata aca 576 Gly Tyr Arg Asp Gly Ala Leu Ile Ala AlaAla Tyr Val Ser Ile Thr 180 185 190 gat tta gca gat tac aat cct aat ttgggt ctt aca aat gaa ggg aat 624 Asp Leu Ala Asp Tyr Asn Pro Asn Leu GlyLeu Thr Asn Glu Gly Asn 195 200 205 ggg gtt gct cat ttt aaa ggt gaa ggttat ata gag ggt gcg caa ggc 672 Gly Val Ala His Phe Lys Gly Glu Gly TyrIle Glu Gly Ala Gln Gly 210 215 220 tta aga agc tac att caa gtt aca gaatat cca gtg gat gat aat ggc 720 Leu Arg Ser Tyr Ile Gln Val Thr Glu TyrPro Val Asp Asp Asn Gly 225 230 235 240 aga cat tcg ata cca aaa act tatata att aaa ggt tca tta gca ccc 768 Arg His Ser Ile Pro Lys Thr Tyr IleIle Lys Gly Ser Leu Ala Pro 245 250 255 aat gtt act tta ata aat gat agaaag gaa ggt aga gga tcc ggt gga 816 Asn Val Thr Leu Ile Asn Asp Arg LysGlu Gly Arg Gly Ser Gly Gly 260 265 270 gct agc atg aca gta tat aac gtaact ttt acc att aaa ttc tat aat 864 Ala Ser Met Thr Val Tyr Asn Val ThrPhe Thr Ile Lys Phe Tyr Asn 275 280 285 gaa ggt gaa tgg ggg ggg cca gaacct tac ggt aag ata tat gca tat 912 Glu Gly Glu Trp Gly Gly Pro Glu ProTyr Gly Lys Ile Tyr Ala Tyr 290 295 300 ctt caa aat cca gat cat aat ttcgaa att tgg tca caa gat aat tgg 960 Leu Gln Asn Pro Asp His Asn Phe GluIle Trp Ser Gln Asp Asn Trp 305 310 315 320 ggg aag gat acg cct gag aaaagt tct cac act caa aca att aaa ata 1008 Gly Lys Asp Thr Pro Glu Lys SerSer His Thr Gln Thr Ile Lys Ile 325 330 335 agt agc cca aca ggg ggg cctata aac caa atg tgt ttt tat ggt gat 1056 Ser Ser Pro Thr Gly Gly Pro IleAsn Gln Met Cys Phe Tyr Gly Asp 340 345 350 gta aaa gaa tac gac gta ggaaat gca gat gat gtt ctc gcc tat cca 1104 Val Lys Glu Tyr Asp Val Gly AsnAla Asp Asp Val Leu Ala Tyr Pro 355 360 365 agt caa aaa gta tgc agt acgcct ggc aca aca ata agg ctt aac gga 1152 Ser Gln Lys Val Cys Ser Thr ProGly Thr Thr Ile Arg Leu Asn Gly 370 375 380 gat gag aaa ggt tct tat atacag att aga tat tcc ttg gcc cca gct 1200 Asp Glu Lys Gly Ser Tyr Ile GlnIle Arg Tyr Ser Leu Ala Pro Ala 385 390 395 400 8 400 PRT Artificialsequence Recombinant fusion protein 8 Met Gly Ile Ile Asn Ile Gln AspGlu Ile Asn Asp Tyr Met Lys Gly 1 5 10 15 Met Tyr Gly Ala Thr Ser ValLys Ser Thr Tyr Asp Pro Ser Phe Lys 20 25 30 Val Phe Asn Glu Ser Val ThrPro Gln Tyr Asp Val Ile Pro Thr Glu 35 40 45 Pro Val Asn Asn His Ile ThrThr Lys Val Ile Asp Asn Pro Gly Thr 50 55 60 Ser Glu Val Thr Ser Thr ValThr Phe Thr Trp Thr Glu Thr Asp Thr 65 70 75 80 Val Thr Ser Ala Val ThrLys Gly Tyr Lys Val Gly Gly Ser Val Ser 85 90 95 Ser Lys Ala Thr Phe LysPhe Ala Phe Val Thr Ser Asp Val Thr Val 100 105 110 Thr Val Ser Ala GluTyr Asn Tyr Ser Thr Thr Glu Thr Thr Thr Lys 115 120 125 Thr Asp Thr ArgThr Trp Thr Asp Ser Thr Thr Val Lys Ala Pro Pro 130 135 140 Arg Thr AsnVal Glu Val Ala Tyr Ile Ile Gln Thr Gly Asn Tyr Asn 145 150 155 160 ValPro Val Asn Val Glu Ser Asp Met Thr Gly Thr Leu Phe Cys Arg 165 170 175Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Ala Tyr Val Ser Ile Thr 180 185190 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Gly Leu Thr Asn Glu Gly Asn 195200 205 Gly Val Ala His Phe Lys Gly Glu Gly Tyr Ile Glu Gly Ala Gln Gly210 215 220 Leu Arg Ser Tyr Ile Gln Val Thr Glu Tyr Pro Val Asp Asp AsnGly 225 230 235 240 Arg His Ser Ile Pro Lys Thr Tyr Ile Ile Lys Gly SerLeu Ala Pro 245 250 255 Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly ArgGly Ser Gly Gly 260 265 270 Ala Ser Met Thr Val Tyr Asn Val Thr Phe ThrIle Lys Phe Tyr Asn 275 280 285 Glu Gly Glu Trp Gly Gly Pro Glu Pro TyrGly Lys Ile Tyr Ala Tyr 290 295 300 Leu Gln Asn Pro Asp His Asn Phe GluIle Trp Ser Gln Asp Asn Trp 305 310 315 320 Gly Lys Asp Thr Pro Glu LysSer Ser His Thr Gln Thr Ile Lys Ile 325 330 335 Ser Ser Pro Thr Gly GlyPro Ile Asn Gln Met Cys Phe Tyr Gly Asp 340 345 350 Val Lys Glu Tyr AspVal Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro 355 360 365 Ser Gln Lys ValCys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly 370 375 380 Asp Glu LysGly Ser Tyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala 385 390 395 400 91188 DNA Artificial sequence Recombinant fusion protein 9 atg gga attatc aac att caa gac gaa att aat gac tac atg aaa ggt 48 Met Gly Ile IleAsn Ile Gln Asp Glu Ile Asn Asp Tyr Met Lys Gly 1 5 10 15 atg tat ggtgca aca tct gtt aaa agc act tat gac ccc tca ttc aaa 96 Met Tyr Gly AlaThr Ser Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 20 25 30 gta ttt aac gaatct gtg aca cct caa tat gat gtg att cca aca gaa 144 Val Phe Asn Glu SerVal Thr Pro Gln Tyr Asp Val Ile Pro Thr Glu 35 40 45 cct gta aat aat catatt act act aaa gta ata gat aat cca ggg act 192 Pro Val Asn Asn His IleThr Thr Lys Val Ile Asp Asn Pro Gly Thr 50 55 60 tca gaa gta acc agt acagta acg ttc aca tgg acg gaa acc gac act 240 Ser Glu Val Thr Ser Thr ValThr Phe Thr Trp Thr Glu Thr Asp Thr 65 70 75 80 gta acc tct gca gtg actaaa ggg tat aaa gtc ggt ggt tca gta agc 288 Val Thr Ser Ala Val Thr LysGly Tyr Lys Val Gly Gly Ser Val Ser 85 90 95 tca aaa gca act ttt aaa tttgct ttt gtt act tct gat gtt act gta 336 Ser Lys Ala Thr Phe Lys Phe AlaPhe Val Thr Ser Asp Val Thr Val 100 105 110 act gta tca gca gaa tat aattat agt aca aca gaa aca aca aca aaa 384 Thr Val Ser Ala Glu Tyr Asn TyrSer Thr Thr Glu Thr Thr Thr Lys 115 120 125 aca gat aca cgc aca tgg acggat tcg acg aca gta aaa gcc cct cca 432 Thr Asp Thr Arg Thr Trp Thr AspSer Thr Thr Val Lys Ala Pro Pro 130 135 140 aga act aat gta gaa gtt gcatat att atc caa act gga aat tat aac 480 Arg Thr Asn Val Glu Val Ala TyrIle Ile Gln Thr Gly Asn Tyr Asn 145 150 155 160 gtt ccg gtt aat gta gagtct gat atg act gga acg cta ttt tgc aga 528 Val Pro Val Asn Val Glu SerAsp Met Thr Gly Thr Leu Phe Cys Arg 165 170 175 ggg tat aga gat ggt gcacta att gca gcg gct tat gtt tct ata aca 576 Gly Tyr Arg Asp Gly Ala LeuIle Ala Ala Ala Tyr Val Ser Ile Thr 180 185 190 gat tta gca gat tac aatcct aat ttg ggt ctt aca aat gaa ggg aat 624 Asp Leu Ala Asp Tyr Asn ProAsn Leu Gly Leu Thr Asn Glu Gly Asn 195 200 205 ggg gtt gct cat ttt aaaggt gaa ggt tat ata gag ggt gcg caa ggc 672 Gly Val Ala His Phe Lys GlyGlu Gly Tyr Ile Glu Gly Ala Gln Gly 210 215 220 tta aga agc tac att caagtt aca gaa tat cca gtg gat gat aat ggc 720 Leu Arg Ser Tyr Ile Gln ValThr Glu Tyr Pro Val Asp Asp Asn Gly 225 230 235 240 aga cat tcg ata ccaaaa act tat ata att aaa ggt tca tta gca ccc 768 Arg His Ser Ile Pro LysThr Tyr Ile Ile Lys Gly Ser Leu Ala Pro 245 250 255 aat gtt act tta ataaat gat aga aag gaa ggt aga ggt gga atg aca 816 Asn Val Thr Leu Ile AsnAsp Arg Lys Glu Gly Arg Gly Gly Met Thr 260 265 270 gta tat aac gta actttt acc att aaa ttc tat aat gaa ggt gaa tgg 864 Val Tyr Asn Val Thr PheThr Ile Lys Phe Tyr Asn Glu Gly Glu Trp 275 280 285 ggg ggg cca gaa ccttac ggt aag ata tat gca tat ctt caa aat cca 912 Gly Gly Pro Glu Pro TyrGly Lys Ile Tyr Ala Tyr Leu Gln Asn Pro 290 295 300 gat cat aat ttc gaaatt tgg tca caa gat aat tgg ggg aag gat acg 960 Asp His Asn Phe Glu IleTrp Ser Gln Asp Asn Trp Gly Lys Asp Thr 305 310 315 320 cct gag aaa agttct cac act caa aca att aaa ata agt agc cca aca 1008 Pro Glu Lys Ser SerHis Thr Gln Thr Ile Lys Ile Ser Ser Pro Thr 325 330 335 ggg ggg cct ataaac caa atg tgt ttt tat ggt gat gta aaa gaa tac 1056 Gly Gly Pro Ile AsnGln Met Cys Phe Tyr Gly Asp Val Lys Glu Tyr 340 345 350 gac gta gga aatgca gat gat gtt ctc gcc tat cca agt caa aaa gta 1104 Asp Val Gly Asn AlaAsp Asp Val Leu Ala Tyr Pro Ser Gln Lys Val 355 360 365 tgc agt acg cctggc aca aca ata agg ctt aac gga gat gag aaa ggt 1152 Cys Ser Thr Pro GlyThr Thr Ile Arg Leu Asn Gly Asp Glu Lys Gly 370 375 380 tct tat ata cagatt aga tat tcc ttg gcc cca gct 1188 Ser Tyr Ile Gln Ile Arg Tyr Ser LeuAla Pro Ala 385 390 395 10 396 PRT Artificial sequence Recombinantfusion protein 10 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp TyrMet Lys Gly 1 5 10 15 Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr AspPro Ser Phe Lys 20 25 30 Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp ValIle Pro Thr Glu 35 40 45 Pro Val Asn Asn His Ile Thr Thr Lys Val Ile AspAsn Pro Gly Thr 50 55 60 Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp ThrGlu Thr Asp Thr 65 70 75 80 Val Thr Ser Ala Val Thr Lys Gly Tyr Lys ValGly Gly Ser Val Ser 85 90 95 Ser Lys Ala Thr Phe Lys Phe Ala Phe Val ThrSer Asp Val Thr Val 100 105 110 Thr Val Ser Ala Glu Tyr Asn Tyr Ser ThrThr Glu Thr Thr Thr Lys 115 120 125 Thr Asp Thr Arg Thr Trp Thr Asp SerThr Thr Val Lys Ala Pro Pro 130 135 140 Arg Thr Asn Val Glu Val Ala TyrIle Ile Gln Thr Gly Asn Tyr Asn 145 150 155 160 Val Pro Val Asn Val GluSer Asp Met Thr Gly Thr Leu Phe Cys Arg 165 170 175 Gly Tyr Arg Asp GlyAla Leu Ile Ala Ala Ala Tyr Val Ser Ile Thr 180 185 190 Asp Leu Ala AspTyr Asn Pro Asn Leu Gly Leu Thr Asn Glu Gly Asn 195 200 205 Gly Val AlaHis Phe Lys Gly Glu Gly Tyr Ile Glu Gly Ala Gln Gly 210 215 220 Leu ArgSer Tyr Ile Gln Val Thr Glu Tyr Pro Val Asp Asp Asn Gly 225 230 235 240Arg His Ser Ile Pro Lys Thr Tyr Ile Ile Lys Gly Ser Leu Ala Pro 245 250255 Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly Arg Gly Gly Met Thr 260265 270 Val Tyr Asn Val Thr Phe Thr Ile Lys Phe Tyr Asn Glu Gly Glu Trp275 280 285 Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala Tyr Leu Gln AsnPro 290 295 300 Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn Trp Gly LysAsp Thr 305 310 315 320 Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys IleSer Ser Pro Thr 325 330 335 Gly Gly Pro Ile Asn Gln Met Cys Phe Tyr GlyAsp Val Lys Glu Tyr 340 345 350 Asp Val Gly Asn Ala Asp Asp Val Leu AlaTyr Pro Ser Gln Lys Val 355 360 365 Cys Ser Thr Pro Gly Thr Thr Ile ArgLeu Asn Gly Asp Glu Lys Gly 370 375 380 Ser Tyr Ile Gln Ile Arg Tyr SerLeu Ala Pro Ala 385 390 395 11 1227 DNA Artificial sequence Recombinantfusion protein 11 atg ggt atc atc aac att caa gat gag att aac aat tacatg aag gaa 48 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn Tyr MetLys Glu 1 5 10 15 gtt tac ggt gct act act gtt aag tct act tac gat ccttct ttc aag 96 Val Tyr Gly Ala Thr Thr Val Lys Ser Thr Tyr Asp Pro SerPhe Lys 20 25 30 gtt ttc aat gaa tct gtt act cct caa ttc act gaa att cctact gaa 144 Val Phe Asn Glu Ser Val Thr Pro Gln Phe Thr Glu Ile Pro ThrGlu 35 40 45 cct gtc aac aac cag ctt act act aag agg gtc gac aat act ggttct 192 Pro Val Asn Asn Gln Leu Thr Thr Lys Arg Val Asp Asn Thr Gly Ser50 55 60 tac cct gtt gaa tct act gtt tct ttc act tgg act gaa act cat act240 Tyr Pro Val Glu Ser Thr Val Ser Phe Thr Trp Thr Glu Thr His Thr 6570 75 80 gaa act tct gct gtt act gaa ggt gtt aag gct ggt act tct att tct288 Glu Thr Ser Ala Val Thr Glu Gly Val Lys Ala Gly Thr Ser Ile Ser 8590 95 act aag caa tct ttc aag ttc ggt ttc gtg aac tct gat gtt act ctt336 Thr Lys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser Asp Val Thr Leu 100105 110 act gtt tct gct gag tac aac tac tct act act aac act act act act384 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asn Thr Thr Thr Thr 115120 125 act gaa act cat act tgg tct gat tct act aag gtt act att cct cct432 Thr Glu Thr His Thr Trp Ser Asp Ser Thr Lys Val Thr Ile Pro Pro 130135 140 aag act tac gtt gaa gct gct tac atc atc cag aat ggt act tac aat480 Lys Thr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn Gly Thr Tyr Asn 145150 155 160 gtt cct gtt aat gtt gaa tgc gat atg tct ggt act ctg ttc tgtcga 528 Val Pro Val Asn Val Glu Cys Asp Met Ser Gly Thr Leu Phe Cys Arg165 170 175 ggt tat cgt gat ggt gct ctt att gct gct gtt tac gtt tct gttgct 576 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Val Tyr Val Ser Val Ala180 185 190 gat ctt gct gat tac aat cct aat ctt aat ctt act aat aag ggtgat 624 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr Asn Lys Gly Asp195 200 205 ggt att gct cat ttc aag ggt tct gga ttc att gaa ggt gct caaggt 672 Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu Gly Ala Gln Gly210 215 220 ctt aga tct gtg atc caa gtt act gaa tac cct ctt gat gat aataag 720 Leu Arg Ser Val Ile Gln Val Thr Glu Tyr Pro Leu Asp Asp Asn Lys225 230 235 240 ggt agg tct act cct att acg tac ctt atc aac ggt tct cttgct cct 768 Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly Ser Leu AlaPro 245 250 255 aat gtt act ctt aag aat tct aat att aag ttc gga tcc ggtgga ggt 816 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe Gly Ser Gly GlyGly 260 265 270 tcc ggt gga ggt tcc ggt gga ggt tcc gct agc atg act gtgtac aat 864 Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ser Met Thr Val TyrAsn 275 280 285 gct act ttc act atc aac ttt tac aat gaa ggt gaa tgg ggtggt cct 912 Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly Glu Trp Gly GlyPro 290 295 300 gaa cct tac ggt tac atc aag gca tac ctt act aat cct gatcat gat 960 Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr Asn Pro Asp HisAsp 305 310 315 320 ttc gag att tgg aag caa gat gat tgg ggt aag tct actcct gag agg 1008 Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys Ser Thr ProGlu Arg 325 330 335 tct act tac act caa act att aag ata tct tct gat actggt tct cct 1056 Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser Asp Thr GlySer Pro 340 345 350 atc aac cag atg tgc ttc tac ggt gac gtc aag gaa tacgat gtc ggc 1104 Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys Glu Tyr AspVal Gly 355 360 365 aac gct gat gat att ctt gct tac cct tct caa aag gtttgc tct act 1152 Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln Lys Val CysSer Thr 370 375 380 cct ggt gtt act gtt agg ctt gat ggt gat gag aag ggttct tac gtt 1200 Pro Gly Val Thr Val Arg Leu Asp Gly Asp Glu Lys Gly SerTyr Val 385 390 395 400 act att aag tac tct ctt act cct gct 1227 Thr IleLys Tyr Ser Leu Thr Pro Ala 405 12 409 PRT Artificial sequenceRecombinant fusion protein 12 Met Gly Ile Ile Asn Ile Gln Asp Glu IleAsn Asn Tyr Met Lys Glu 1 5 10 15 Val Tyr Gly Ala Thr Thr Val Lys SerThr Tyr Asp Pro Ser Phe Lys 20 25 30 Val Phe Asn Glu Ser Val Thr Pro GlnPhe Thr Glu Ile Pro Thr Glu 35 40 45 Pro Val Asn Asn Gln Leu Thr Thr LysArg Val Asp Asn Thr Gly Ser 50 55 60 Tyr Pro Val Glu Ser Thr Val Ser PheThr Trp Thr Glu Thr His Thr 65 70 75 80 Glu Thr Ser Ala Val Thr Glu GlyVal Lys Ala Gly Thr Ser Ile Ser 85 90 95 Thr Lys Gln Ser Phe Lys Phe GlyPhe Val Asn Ser Asp Val Thr Leu 100 105 110 Thr Val Ser Ala Glu Tyr AsnTyr Ser Thr Thr Asn Thr Thr Thr Thr 115 120 125 Thr Glu Thr His Thr TrpSer Asp Ser Thr Lys Val Thr Ile Pro Pro 130 135 140 Lys Thr Tyr Val GluAla Ala Tyr Ile Ile Gln Asn Gly Thr Tyr Asn 145 150 155 160 Val Pro ValAsn Val Glu Cys Asp Met Ser Gly Thr Leu Phe Cys Arg 165 170 175 Gly TyrArg Asp Gly Ala Leu Ile Ala Ala Val Tyr Val Ser Val Ala 180 185 190 AspLeu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr Asn Lys Gly Asp 195 200 205Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu Gly Ala Gln Gly 210 215220 Leu Arg Ser Val Ile Gln Val Thr Glu Tyr Pro Leu Asp Asp Asn Lys 225230 235 240 Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly Ser Leu AlaPro 245 250 255 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe Gly Ser GlyGly Gly 260 265 270 Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ser Met ThrVal Tyr Asn 275 280 285 Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly GluTrp Gly Gly Pro 290 295 300 Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu ThrAsn Pro Asp His Asp 305 310 315 320 Phe Glu Ile Trp Lys Gln Asp Asp TrpGly Lys Ser Thr Pro Glu Arg 325 330 335 Ser Thr Tyr Thr Gln Thr Ile LysIle Ser Ser Asp Thr Gly Ser Pro 340 345 350 Ile Asn Gln Met Cys Phe TyrGly Asp Val Lys Glu Tyr Asp Val Gly 355 360 365 Asn Ala Asp Asp Ile LeuAla Tyr Pro Ser Gln Lys Val Cys Ser Thr 370 375 380 Pro Gly Val Thr ValArg Leu Asp Gly Asp Glu Lys Gly Ser Tyr Val 385 390 395 400 Thr Ile LysTyr Ser Leu Thr Pro Ala 405 13 2397 DNA Artificial sequence Recombinantfusion protein 13 atg aca gta tat aac gta act ttt acc att aaa ttc tataat gaa ggt 48 Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe Tyr AsnGlu Gly 1 5 10 15 gaa tgg ggg ggg cca gaa cct tac ggt aag ata tat gcatac ctt caa 96 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala TyrLeu Gln 20 25 30 aat cca gat cat aat ttc gaa att tgg tca caa gat aat tggggg aag 144 Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn Trp GlyLys 35 40 45 gat acg cct gag aaa agt tct cac act caa aca att aaa ata agtagc 192 Asp Thr Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys Ile Ser Ser50 55 60 cca aca ggg ggg cct ata aac caa atg tgt ttt tat ggt gat gta aaa240 Pro Thr Gly Gly Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 6570 75 80 gaa tac gac gta gga aat gca gat gat gtt ctc gcc tat cca agt caa288 Glu Tyr Asp Val Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro Ser Gln 8590 95 aaa gta tgc agt acg cct ggc aca aca ata agg ctt aac gga gat gag336 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu 100105 110 aaa ggt tct tat ata cag att aga tat tcc ttg gcc cca gct gga tcc384 Lys Gly Ser Tyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala Gly Ser 115120 125 ggt gga gct agc atg gga att atc aac att caa gac gaa att aat gac432 Gly Gly Ala Ser Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp 130135 140 tac atg aaa ggt atg tat ggt gca aca tct gtt aaa agc act tat gac480 Tyr Met Lys Gly Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp 145150 155 160 ccc tca ttc aaa gta ttt aac gaa tct gtg aca cct caa tat gatgtg 528 Pro Ser Phe Lys Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp Val165 170 175 att cca aca gaa cct gta aat aat cat att act act aaa gta atagat 576 Ile Pro Thr Glu Pro Val Asn Asn His Ile Thr Thr Lys Val Ile Asp180 185 190 aat cca ggg act tca gaa gta acc agt aca gta acg ttc aca tggacg 624 Asn Pro Gly Thr Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr195 200 205 gaa acc gac act gta acc tct gca gtg act aaa ggg tat aaa gtcggt 672 Glu Thr Asp Thr Val Thr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly210 215 220 ggt tca gta agc tca aaa gca act ttt aaa ttt gct ttt gtt acttct 720 Gly Ser Val Ser Ser Lys Ala Thr Phe Lys Phe Ala Phe Val Thr Ser225 230 235 240 gat gtt act gta act gta tca gca gaa tat aat tat agt acaaca gaa 768 Asp Val Thr Val Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr ThrGlu 245 250 255 aca aca aca aaa aca gat aca cgc aca tgg acg gat tcg acgaca gta 816 Thr Thr Thr Lys Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr ThrVal 260 265 270 aaa gcc cct cca aga act aat gta gaa gtt gca tat att atccaa act 864 Lys Ala Pro Pro Arg Thr Asn Val Glu Val Ala Tyr Ile Ile GlnThr 275 280 285 gga aat tat aac gtt ccg gtt aat gta gag tct gat atg actgga acg 912 Gly Asn Tyr Asn Val Pro Val Asn Val Glu Ser Asp Met Thr GlyThr 290 295 300 cta ttt tgc aga ggg tat aga gat ggt gca cta att gca gcggct tat 960 Leu Phe Cys Arg Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala AlaTyr 305 310 315 320 gtt tct ata aca gat tta gca gat tac aat cct aat ttgggt ctt aca 1008 Val Ser Ile Thr Asp Leu Ala Asp Tyr Asn Pro Asn Leu GlyLeu Thr 325 330 335 aat gaa ggg aat ggg gtt gct cat ttt aaa ggt gaa ggttat ata gag 1056 Asn Glu Gly Asn Gly Val Ala His Phe Lys Gly Glu Gly TyrIle Glu 340 345 350 ggt gcg caa ggc tta aga agc tac att caa gtt aca gaatat cca gtg 1104 Gly Ala Gln Gly Leu Arg Ser Tyr Ile Gln Val Thr Glu TyrPro Val 355 360 365 gat gat aat ggc aga cat tcg ata cca aaa act tat ataatt aaa ggt 1152 Asp Asp Asn Gly Arg His Ser Ile Pro Lys Thr Tyr Ile IleLys Gly 370 375 380 tca tta gca ccc aat gtt act tta ata aat gat aga aaggaa ggt 1197 Ser Leu Ala Pro Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly385 390 395 agaatgggaa ttattaatat ccaagatgaa attaataatt acatgaaagaggtatatggt 1257 gcaacaactg ttaaaagcac atacgatccc tcattcaaag tatttaatgaatctgtgaca 1317 ccccaattca ctgaaattcc aacagaacct gtaaataatc aattaactacaaaaagagta 1377 gataatacgg gtagttaccc agtagaaagt actgtatcgt tcacatggacggaaacccat 1437 acagaaacaa gtgcagtaac tgagggagtg aaagccggca cctcaataagtactaaacaa 1497 tcttttaaat ttggttttgt taactctgat gttactttaa cggtatcagcagaatataat 1557 tatagtacaa caaatacaac tacaacaaca gaaacacaca cctggtcagattcaacaaaa 1617 gtaactattc ctcccaaaac ttatgtggag gctgcataca ttatccaaaatggaacatat 1677 aatgttccgg ttaatgtaga atgtgatatg agtggaactt tattttgtagagggtataga 1737 gatggtgcgc ttattgcagc agtttatgtt tctgtagcgg atttagcagattacaatcca 1797 aatttaaatc ttacaaataa aggggatgga attgctcact ttaaaggttcgggttttata 1857 gagggtgcac aaggcttgcg aagcattatt caggttacag aatatccactagatgataat 1917 aaaggtcgct cgacaccaat aacttattta ataaatggtt cattagcaccaaatgttaca 1977 ttaaaaaata gcaacataaa atttggatcc ggtggagcta gcatgacagtatataacgca 2037 actttcacca ttaatttcta taatgaagga gaatgggggg ggccagaaccatatggttat 2097 ataaaagcat atcttacaaa tccagatcat gattttgaaa tttggaaacaagatgattgg 2157 gggaaaagta ctcctgagag aagtacttat acgcaaacga ttaaaataagtagcgacact 2217 ggttccccta taaaccaaat gtgtttttat ggtgatgtga aagaatacgacgtaggaaat 2277 gcagatgata ttctcgctta tccaagtcaa aaagtatgca gtacacctggtgtaacagta 2337 cgacttgatg gcgatgagaa aggttcttat gtgacaatta agtattccttgactccagca 2397 14 399 PRT Artificial sequence Recombinant fusionprotein 14 Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe Tyr Asn GluGly 1 5 10 15 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala TyrLeu Gln 20 25 30 Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn TrpGly Lys 35 40 45 Asp Thr Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys IleSer Ser 50 55 60 Pro Thr Gly Gly Pro Ile Asn Gln Met Cys Phe Tyr Gly AspVal Lys 65 70 75 80 Glu Tyr Asp Val Gly Asn Ala Asp Asp Val Leu Ala TyrPro Ser Gln 85 90 95 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu AsnGly Asp Glu 100 105 110 Lys Gly Ser Tyr Ile Gln Ile Arg Tyr Ser Leu AlaPro Ala Gly Ser 115 120 125 Gly Gly Ala Ser Met Gly Ile Ile Asn Ile GlnAsp Glu Ile Asn Asp 130 135 140 Tyr Met Lys Gly Met Tyr Gly Ala Thr SerVal Lys Ser Thr Tyr Asp 145 150 155 160 Pro Ser Phe Lys Val Phe Asn GluSer Val Thr Pro Gln Tyr Asp Val 165 170 175 Ile Pro Thr Glu Pro Val AsnAsn His Ile Thr Thr Lys Val Ile Asp 180 185 190 Asn Pro Gly Thr Ser GluVal Thr Ser Thr Val Thr Phe Thr Trp Thr 195 200 205 Glu Thr Asp Thr ValThr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly 210 215 220 Gly Ser Val SerSer Lys Ala Thr Phe Lys Phe Ala Phe Val Thr Ser 225 230 235 240 Asp ValThr Val Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Glu 245 250 255 ThrThr Thr Lys Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val 260 265 270Lys Ala Pro Pro Arg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr 275 280285 Gly Asn Tyr Asn Val Pro Val Asn Val Glu Ser Asp Met Thr Gly Thr 290295 300 Leu Phe Cys Arg Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Ala Tyr305 310 315 320 Val Ser Ile Thr Asp Leu Ala Asp Tyr Asn Pro Asn Leu GlyLeu Thr 325 330 335 Asn Glu Gly Asn Gly Val Ala His Phe Lys Gly Glu GlyTyr Ile Glu 340 345 350 Gly Ala Gln Gly Leu Arg Ser Tyr Ile Gln Val ThrGlu Tyr Pro Val 355 360 365 Asp Asp Asn Gly Arg His Ser Ile Pro Lys ThrTyr Ile Ile Lys Gly 370 375 380 Ser Leu Ala Pro Asn Val Thr Leu Ile AsnAsp Arg Lys Glu Gly 385 390 395 15 1197 DNA Artificial sequenceRecombinant fusion protein 15 atg ggt atc atc aac att caa gat gag attaac aat tac atg aag gaa 48 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile AsnAsn Tyr Met Lys Glu 1 5 10 15 gtt tac ggt gct act act gtt aag tct acttac gat cct tct ttc aag 96 Val Tyr Gly Ala Thr Thr Val Lys Ser Thr TyrAsp Pro Ser Phe Lys 20 25 30 gtt ttc aat gaa tct gtt act cct caa ttc actgaa att cct act gaa 144 Val Phe Asn Glu Ser Val Thr Pro Gln Phe Thr GluIle Pro Thr Glu 35 40 45 cct gtc aac aac cag ctt act act aag agg gtc gacaat act ggt tct 192 Pro Val Asn Asn Gln Leu Thr Thr Lys Arg Val Asp AsnThr Gly Ser 50 55 60 tac cct gtt gaa tct act gtt tct ttc act tgg act gaaact cat act 240 Tyr Pro Val Glu Ser Thr Val Ser Phe Thr Trp Thr Glu ThrHis Thr 65 70 75 80 gaa act tct gct gtt act gaa ggt gtt aag gct ggt acttct att tct 288 Glu Thr Ser Ala Val Thr Glu Gly Val Lys Ala Gly Thr SerIle Ser 85 90 95 act aag caa tct ttc aag ttc ggt ttc gtg aac tct gat gttact ctt 336 Thr Lys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser Asp Val ThrLeu 100 105 110 act gtt tct gct gag tac aac tac tct act act aac act actact act 384 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asn Thr Thr ThrThr 115 120 125 act gaa act cat act tgg tct gat tct act aag gtt act attcct cct 432 Thr Glu Thr His Thr Trp Ser Asp Ser Thr Lys Val Thr Ile ProPro 130 135 140 aag act tac gtt gaa gct gct tac atc atc cag aat ggt acttac aat 480 Lys Thr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn Gly Thr TyrAsn 145 150 155 160 gtt cct gtt aat gtt gaa tgc gat atg tct ggt act ctgttc tgt cga 528 Val Pro Val Asn Val Glu Cys Asp Met Ser Gly Thr Leu PheCys Arg 165 170 175 ggt tat cgt gat ggt gct ctt att gct gct gtt tac gtttct gtt gct 576 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Val Tyr Val SerVal Ala 180 185 190 gat ctt gct gat tac aat cct aat ctt aat ctt act aataag ggt gat 624 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr Asn LysGly Asp 195 200 205 ggt att gct cat ttc aag ggt tct gga ttc att gaa ggtgct caa ggt 672 Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu Gly AlaGln Gly 210 215 220 ctt aga tct gtg atc caa gtt act gaa tac cct ctt gatgat aat aag 720 Leu Arg Ser Val Ile Gln Val Thr Glu Tyr Pro Leu Asp AspAsn Lys 225 230 235 240 ggt agg tct act cct att acg tac ctt atc aac ggttct ctt gct cct 768 Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly SerLeu Ala Pro 245 250 255 aat gtt act ctt aag aat tct aat att aag ttc ggatcc ggt gga gct 816 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe Gly SerGly Gly Ala 260 265 270 agc atg act gtg tac aat gct act ttc act atc aacttt tac aat gaa 864 Ser Met Thr Val Tyr Asn Ala Thr Phe Thr Ile Asn PheTyr Asn Glu 275 280 285 ggt gaa tgg ggt ggt cct gaa cct tac ggt tac atcaag gca tac ctt 912 Gly Glu Trp Gly Gly Pro Glu Pro Tyr Gly Tyr Ile LysAla Tyr Leu 290 295 300 act aat cct gat cat gat ttc gag att tgg aag caagat gat tgg ggt 960 Thr Asn Pro Asp His Asp Phe Glu Ile Trp Lys Gln AspAsp Trp Gly 305 310 315 320 aag tct act cct gag agg tct act tac act caaact att aag ata tct 1008 Lys Ser Thr Pro Glu Arg Ser Thr Tyr Thr Gln ThrIle Lys Ile Ser 325 330 335 tct gat act ggt tct cct atc aac cag atg tgcttc tac ggt gac gtc 1056 Ser Asp Thr Gly Ser Pro Ile Asn Gln Met Cys PheTyr Gly Asp Val 340 345 350 aag gaa tac gat gtc ggc aac gct gat gat attctt gct tac cct tct 1104 Lys Glu Tyr Asp Val Gly Asn Ala Asp Asp Ile LeuAla Tyr Pro Ser 355 360 365 caa aag gtt tgc tct act cct ggt gtt act gttagg ctt gat ggt gat 1152 Gln Lys Val Cys Ser Thr Pro Gly Val Thr Val ArgLeu Asp Gly Asp 370 375 380 gag aag ggt tct tac gtt act att aag tac tctctt act cct gct 1197 Glu Lys Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu ThrPro Ala 385 390 395 16 399 PRT Artificial sequence Recombinant fusionprotein 16 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn Tyr Met LysGlu 1 5 10 15 Val Tyr Gly Ala Thr Thr Val Lys Ser Thr Tyr Asp Pro SerPhe Lys 20 25 30 Val Phe Asn Glu Ser Val Thr Pro Gln Phe Thr Glu Ile ProThr Glu 35 40 45 Pro Val Asn Asn Gln Leu Thr Thr Lys Arg Val Asp Asn ThrGly Ser 50 55 60 Tyr Pro Val Glu Ser Thr Val Ser Phe Thr Trp Thr Glu ThrHis Thr 65 70 75 80 Glu Thr Ser Ala Val Thr Glu Gly Val Lys Ala Gly ThrSer Ile Ser 85 90 95 Thr Lys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser AspVal Thr Leu 100 105 110 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr AsnThr Thr Thr Thr 115 120 125 Thr Glu Thr His Thr Trp Ser Asp Ser Thr LysVal Thr Ile Pro Pro 130 135 140 Lys Thr Tyr Val Glu Ala Ala Tyr Ile IleGln Asn Gly Thr Tyr Asn 145 150 155 160 Val Pro Val Asn Val Glu Cys AspMet Ser Gly Thr Leu Phe Cys Arg 165 170 175 Gly Tyr Arg Asp Gly Ala LeuIle Ala Ala Val Tyr Val Ser Val Ala 180 185 190 Asp Leu Ala Asp Tyr AsnPro Asn Leu Asn Leu Thr Asn Lys Gly Asp 195 200 205 Gly Ile Ala His PheLys Gly Ser Gly Phe Ile Glu Gly Ala Gln Gly 210 215 220 Leu Arg Ser ValIle Gln Val Thr Glu Tyr Pro Leu Asp Asp Asn Lys 225 230 235 240 Gly ArgSer Thr Pro Ile Thr Tyr Leu Ile Asn Gly Ser Leu Ala Pro 245 250 255 AsnVal Thr Leu Lys Asn Ser Asn Ile Lys Phe Gly Ser Gly Gly Ala 260 265 270Ser Met Thr Val Tyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu 275 280285 Gly Glu Trp Gly Gly Pro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu 290295 300 Thr Asn Pro Asp His Asp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly305 310 315 320 Lys Ser Thr Pro Glu Arg Ser Thr Tyr Thr Gln Thr Ile LysIle Ser 325 330 335 Ser Asp Thr Gly Ser Pro Ile Asn Gln Met Cys Phe TyrGly Asp Val 340 345 350 Lys Glu Tyr Asp Val Gly Asn Ala Asp Asp Ile LeuAla Tyr Pro Ser 355 360 365 Gln Lys Val Cys Ser Thr Pro Gly Val Thr ValArg Leu Asp Gly Asp 370 375 380 Glu Lys Gly Ser Tyr Val Thr Ile Lys TyrSer Leu Thr Pro Ala 385 390 395 17 1239 DNA Artificial sequenceRecombinant fusion protein 17 atg ggt atc atc aac att caa gat gag attaac aat tac atg aag gaa 48 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile AsnAsn Tyr Met Lys Glu 1 5 10 15 gtt tac ggt gct act act gtt aag tct acttac gat cct tct ttc aag 96 Val Tyr Gly Ala Thr Thr Val Lys Ser Thr TyrAsp Pro Ser Phe Lys 20 25 30 gtt ttc aat gaa tct gtt act cct caa ttc actgaa att cct act gaa 144 Val Phe Asn Glu Ser Val Thr Pro Gln Phe Thr GluIle Pro Thr Glu 35 40 45 cct gtc aac aac cag ctt act act aag agg gtc gacaat act ggt tct 192 Pro Val Asn Asn Gln Leu Thr Thr Lys Arg Val Asp AsnThr Gly Ser 50 55 60 tac cct gtt gaa tct act gtt tct ttc act tgg act gaaact cat act 240 Tyr Pro Val Glu Ser Thr Val Ser Phe Thr Trp Thr Glu ThrHis Thr 65 70 75 80 gaa act tct gct gtt act gaa ggt gtt aag gct ggt acttct att tct 288 Glu Thr Ser Ala Val Thr Glu Gly Val Lys Ala Gly Thr SerIle Ser 85 90 95 act aag caa tct ttc aag ttc ggt ttc gtg aac tct gat gttact ctt 336 Thr Lys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser Asp Val ThrLeu 100 105 110 act gtt tct gct gag tac aac tac tct act act aac act actact act 384 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asn Thr Thr ThrThr 115 120 125 act gaa act cat act tgg tct gat tct act aag gtt act attcct cct 432 Thr Glu Thr His Thr Trp Ser Asp Ser Thr Lys Val Thr Ile ProPro 130 135 140 aag act tac gtt gaa gct gct tac atc atc cag aat ggt acttac aat 480 Lys Thr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn Gly Thr TyrAsn 145 150 155 160 gtt cct gtt aat gtt gaa tgc gat atg tct ggt act ctgttc tgt cga 528 Val Pro Val Asn Val Glu Cys Asp Met Ser Gly Thr Leu PheCys Arg 165 170 175 ggt tat cgt gat ggt gct ctt att gct gct gtt tac gtttct gtt gct 576 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Val Tyr Val SerVal Ala 180 185 190 gat ctt gct gat tac aat cct aat ctt aat ctt act aataag ggt gat 624 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr Asn LysGly Asp 195 200 205 ggt att gct cat ttc aag ggt tct gga ttc att gaa ggtgct caa ggt 672 Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu Gly AlaGln Gly 210 215 220 ctt aga tct gtg atc caa gtt act gaa tac cct ctt gatgat aat aag 720 Leu Arg Ser Val Ile Gln Val Thr Glu Tyr Pro Leu Asp AspAsn Lys 225 230 235 240 ggt agg tct act cct att acg tac ctt atc aac ggttct ctt gct cct 768 Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly SerLeu Ala Pro 245 250 255 aat gtt act ctt aag aat tct aat att aag ttc ggatcc cca gct ttg 816 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe Gly SerPro Ala Leu 260 265 270 ctt aag gag gct cca aga gct gag gaa gag ttg ccacca gct agc atg 864 Leu Lys Glu Ala Pro Arg Ala Glu Glu Glu Leu Pro ProAla Ser Met 275 280 285 act gtg tac aat gct act ttc act atc aac ttt tacaat gaa ggt gaa 912 Thr Val Tyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr AsnGlu Gly Glu 290 295 300 tgg ggt ggt cct gaa cct tac ggt tac atc aag gcatac ctt act aat 960 Trp Gly Gly Pro Glu Pro Tyr Gly Tyr Ile Lys Ala TyrLeu Thr Asn 305 310 315 320 cct gat cat gat ttc gag att tgg aag caa gatgat tgg ggt aag tct 1008 Pro Asp His Asp Phe Glu Ile Trp Lys Gln Asp AspTrp Gly Lys Ser 325 330 335 act cct gag agg tct act tac act caa act attaag ata tct tct gat 1056 Thr Pro Glu Arg Ser Thr Tyr Thr Gln Thr Ile LysIle Ser Ser Asp 340 345 350 act ggt tct cct atc aac cag atg tgc ttc tacggt gac gtc aag gaa 1104 Thr Gly Ser Pro Ile Asn Gln Met Cys Phe Tyr GlyAsp Val Lys Glu 355 360 365 tac gat gtc ggc aac gct gat gat att ctt gcttac cct tct caa aag 1152 Tyr Asp Val Gly Asn Ala Asp Asp Ile Leu Ala TyrPro Ser Gln Lys 370 375 380 gtt tgc tct act cct ggt gtt act gtt agg cttgat ggt gat gag aag 1200 Val Cys Ser Thr Pro Gly Val Thr Val Arg Leu AspGly Asp Glu Lys 385 390 395 400 ggt tct tac gtt act att aag tac tct cttact cct gct 1239 Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala 405410 18 413 PRT Artificial sequence Recombinant fusion protein 18 Met GlyIle Ile Asn Ile Gln Asp Glu Ile Asn Asn Tyr Met Lys Glu 1 5 10 15 ValTyr Gly Ala Thr Thr Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 20 25 30 ValPhe Asn Glu Ser Val Thr Pro Gln Phe Thr Glu Ile Pro Thr Glu 35 40 45 ProVal Asn Asn Gln Leu Thr Thr Lys Arg Val Asp Asn Thr Gly Ser 50 55 60 TyrPro Val Glu Ser Thr Val Ser Phe Thr Trp Thr Glu Thr His Thr 65 70 75 80Glu Thr Ser Ala Val Thr Glu Gly Val Lys Ala Gly Thr Ser Ile Ser 85 90 95Thr Lys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser Asp Val Thr Leu 100 105110 Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asn Thr Thr Thr Thr 115120 125 Thr Glu Thr His Thr Trp Ser Asp Ser Thr Lys Val Thr Ile Pro Pro130 135 140 Lys Thr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn Gly Thr TyrAsn 145 150 155 160 Val Pro Val Asn Val Glu Cys Asp Met Ser Gly Thr LeuPhe Cys Arg 165 170 175 Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Val TyrVal Ser Val Ala 180 185 190 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Asn LeuThr Asn Lys Gly Asp 195 200 205 Gly Ile Ala His Phe Lys Gly Ser Gly PheIle Glu Gly Ala Gln Gly 210 215 220 Leu Arg Ser Val Ile Gln Val Thr GluTyr Pro Leu Asp Asp Asn Lys 225 230 235 240 Gly Arg Ser Thr Pro Ile ThrTyr Leu Ile Asn Gly Ser Leu Ala Pro 245 250 255 Asn Val Thr Leu Lys AsnSer Asn Ile Lys Phe Gly Ser Pro Ala Leu 260 265 270 Leu Lys Glu Ala ProArg Ala Glu Glu Glu Leu Pro Pro Ala Ser Met 275 280 285 Thr Val Tyr AsnAla Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly Glu 290 295 300 Trp Gly GlyPro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr Asn 305 310 315 320 ProAsp His Asp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys Ser 325 330 335Thr Pro Glu Arg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser Asp 340 345350 Thr Gly Ser Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys Glu 355360 365 Tyr Asp Val Gly Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln Lys370 375 380 Val Cys Ser Thr Pro Gly Val Thr Val Arg Leu Asp Gly Asp GluLys 385 390 395 400 Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala405 410 19 1197 DNA Artificial sequence Recombinant fusion protein 19atg aca gta tat aac gca act ttc acc att aat ttc tat aat gaa gga 48 MetThr Val Tyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15gaa tgg ggg ggg cca gaa cca tat ggt tat ata aaa gca tat ctt aca 96 GluTrp Gly Gly Pro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30 aatcca gat cat gat ttt gaa att tgg aaa caa gat gat tgg ggg aaa 144 Asn ProAsp His Asp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45 agt actcct gag aga agt act tat acg caa acg att aaa ata agt agc 192 Ser Thr ProGlu Arg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 gac act ggttcc cct ata aac caa atg tgt ttt tat ggt gat gtg aaa 240 Asp Thr Gly SerPro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 gaa tac gacgta gga aat gca gat gat att ctc gct tat cca agt caa 288 Glu Tyr Asp ValGly Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85 90 95 aaa gta tgc agtaca cct ggt gta aca gta cga ctt gat ggc gat gag 336 Lys Val Cys Ser ThrPro Gly Val Thr Val Arg Leu Asp Gly Asp Glu 100 105 110 aaa ggt tct tatgtg aca att aag tat tcc ttg act cca gca gga tcc 384 Lys Gly Ser Tyr ValThr Ile Lys Tyr Ser Leu Thr Pro Ala Gly Ser 115 120 125 ggt gga gct agcatg gga att att aat atc caa gat gaa att aat aat 432 Gly Gly Ala Ser MetGly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn 130 135 140 tac atg aaa gaggta tat ggt gca aca act gtt aaa agc aca tac gat 480 Tyr Met Lys Glu ValTyr Gly Ala Thr Thr Val Lys Ser Thr Tyr Asp 145 150 155 160 ccc tca ttcaaa gta ttt aat gaa tct gtg aca ccc caa ttc act gaa 528 Pro Ser Phe LysVal Phe Asn Glu Ser Val Thr Pro Gln Phe Thr Glu 165 170 175 att cca acagaa cct gta aat aat caa tta act aca aaa aga gta gat 576 Ile Pro Thr GluPro Val Asn Asn Gln Leu Thr Thr Lys Arg Val Asp 180 185 190 aat acg ggtagt tac cca gta gaa agt act gta tcg ttc aca tgg acg 624 Asn Thr Gly SerTyr Pro Val Glu Ser Thr Val Ser Phe Thr Trp Thr 195 200 205 gaa acc cataca gaa aca agt gca gta act gag gga gtg aaa gcc ggc 672 Glu Thr His ThrGlu Thr Ser Ala Val Thr Glu Gly Val Lys Ala Gly 210 215 220 acc tca ataagt act aaa caa tct ttt aaa ttt ggt ttt gtt aac tct 720 Thr Ser Ile SerThr Lys Gln Ser Phe Lys Phe Gly Phe Val Asn Ser 225 230 235 240 gat gttact tta acg gta tca gca gaa tat aat tat agt aca aca aat 768 Asp Val ThrLeu Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asn 245 250 255 aca actaca aca aca gaa aca cac acc tgg tca gat tca aca aaa gta 816 Thr Thr ThrThr Thr Glu Thr His Thr Trp Ser Asp Ser Thr Lys Val 260 265 270 act attcct ccc aaa act tat gtg gag gct gca tac att atc caa aat 864 Thr Ile ProPro Lys Thr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn 275 280 285 gga acatat aat gtt ccg gtt aat gta gaa tgt gat atg agt gga act 912 Gly Thr TyrAsn Val Pro Val Asn Val Glu Cys Asp Met Ser Gly Thr 290 295 300 tta ttttgt aga ggg tat aga gat ggt gcg ctt att gca gca gtt tat 960 Leu Phe CysArg Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Val Tyr 305 310 315 320 gtttct gta gcg gat tta gca gat tac aat cca aat tta aat ctt aca 1008 Val SerVal Ala Asp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr 325 330 335 aataaa ggg gat gga att gct cac ttt aaa ggt tcg ggt ttt ata gag 1056 Asn LysGly Asp Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu 340 345 350 ggtgca caa ggc ttg cga agc att att cag gtt aca gaa tat cca cta 1104 Gly AlaGln Gly Leu Arg Ser Ile Ile Gln Val Thr Glu Tyr Pro Leu 355 360 365 gatgat aat aaa ggt cgc tcg aca cca ata act tat tta ata aat ggt 1152 Asp AspAsn Lys Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly 370 375 380 tcatta gca cca aat gtt aca tta aaa aat agc aac ata aaa ttt 1197 Ser Leu AlaPro Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe 385 390 395 20 399 PRTArtificial sequence Recombinant fusion protein 20 Met Thr Val Tyr AsnAla Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15 Glu Trp Gly GlyPro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30 Asn Pro Asp HisAsp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45 Ser Thr Pro GluArg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 Asp Thr Gly SerPro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 Glu Tyr AspVal Gly Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85 90 95 Lys Val CysSer Thr Pro Gly Val Thr Val Arg Leu Asp Gly Asp Glu 100 105 110 Lys GlySer Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala Gly Ser 115 120 125 GlyGly Ala Ser Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn 130 135 140Tyr Met Lys Glu Val Tyr Gly Ala Thr Thr Val Lys Ser Thr Tyr Asp 145 150155 160 Pro Ser Phe Lys Val Phe Asn Glu Ser Val Thr Pro Gln Phe Thr Glu165 170 175 Ile Pro Thr Glu Pro Val Asn Asn Gln Leu Thr Thr Lys Arg ValAsp 180 185 190 Asn Thr Gly Ser Tyr Pro Val Glu Ser Thr Val Ser Phe ThrTrp Thr 195 200 205 Glu Thr His Thr Glu Thr Ser Ala Val Thr Glu Gly ValLys Ala Gly 210 215 220 Thr Ser Ile Ser Thr Lys Gln Ser Phe Lys Phe GlyPhe Val Asn Ser 225 230 235 240 Asp Val Thr Leu Thr Val Ser Ala Glu TyrAsn Tyr Ser Thr Thr Asn 245 250 255 Thr Thr Thr Thr Thr Glu Thr His ThrTrp Ser Asp Ser Thr Lys Val 260 265 270 Thr Ile Pro Pro Lys Thr Tyr ValGlu Ala Ala Tyr Ile Ile Gln Asn 275 280 285 Gly Thr Tyr Asn Val Pro ValAsn Val Glu Cys Asp Met Ser Gly Thr 290 295 300 Leu Phe Cys Arg Gly TyrArg Asp Gly Ala Leu Ile Ala Ala Val Tyr 305 310 315 320 Val Ser Val AlaAsp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr 325 330 335 Asn Lys GlyAsp Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu 340 345 350 Gly AlaGln Gly Leu Arg Ser Ile Ile Gln Val Thr Glu Tyr Pro Leu 355 360 365 AspAsp Asn Lys Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly 370 375 380Ser Leu Ala Pro Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe 385 390 39521 1197 DNA Artificial sequence Recombinant fusion protein 21 atg actgtg tac aat gct act ttc act atc aac ttt tac aat gaa ggt 48 Met Thr ValTyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15 gaa tggggt ggt cct gaa cct tac ggt tac atc aag gca tac ctt act 96 Glu Trp GlyGly Pro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30 aat cct gatcat gat ttc gag att tgg aag caa gat gat tgg ggt aag 144 Asn Pro Asp HisAsp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45 tct act cct gagagg tct act tac act caa act att aag ata tct tct 192 Ser Thr Pro Glu ArgSer Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 gat act ggt tct cctatc aac cag atg tgc ttc tac ggt gac gtc aag 240 Asp Thr Gly Ser Pro IleAsn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 gaa tac gat gtc ggcaac gct gat gat att ctt gct tac cct tct caa 288 Glu Tyr Asp Val Gly AsnAla Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85 90 95 aag gtt tgc tct act cctggt gtt act gtt agg ctt gat ggt gat gag 336 Lys Val Cys Ser Thr Pro GlyVal Thr Val Arg Leu Asp Gly Asp Glu 100 105 110 aag ggt tct tac gtt actatt aag tac tct ctt act cct gct gga tcc 384 Lys Gly Ser Tyr Val Thr IleLys Tyr Ser Leu Thr Pro Ala Gly Ser 115 120 125 ggt gga gct agc atg ggtatc atc aac att caa gat gag att aac aat 432 Gly Gly Ala Ser Met Gly IleIle Asn Ile Gln Asp Glu Ile Asn Asn 130 135 140 tac atg aag gaa gtt tacggt gct act act gtt aag tct act tac gat 480 Tyr Met Lys Glu Val Tyr GlyAla Thr Thr Val Lys Ser Thr Tyr Asp 145 150 155 160 cct tct ttc aag gttttc aat gaa tct gtt act cct caa ttc act gaa 528 Pro Ser Phe Lys Val PheAsn Glu Ser Val Thr Pro Gln Phe Thr Glu 165 170 175 att cct act gaa cctgtc aac aac cag ctt act act aag agg gtc gac 576 Ile Pro Thr Glu Pro ValAsn Asn Gln Leu Thr Thr Lys Arg Val Asp 180 185 190 aat act ggt tct taccct gtt gaa tct act gtt tct tta act tgg act 624 Asn Thr Gly Ser Tyr ProVal Glu Ser Thr Val Ser Leu Thr Trp Thr 195 200 205 gaa act cat act gaaact tct gct gtt act gaa ggt gtt aag gct ggt 672 Glu Thr His Thr Glu ThrSer Ala Val Thr Glu Gly Val Lys Ala Gly 210 215 220 act tct att tct actaag caa tct ttc aag ttc ggt ttc gtg aac tct 720 Thr Ser Ile Ser Thr LysGln Ser Phe Lys Phe Gly Phe Val Asn Ser 225 230 235 240 gat gtt act cttact gtt tct gct gag tac aac tac tct act act aac 768 Asp Val Thr Leu ThrVal Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Asn 245 250 255 act act act actact gaa act cat act tgg tct gat tct act aag gtt 816 Thr Thr Thr Thr ThrGlu Thr His Thr Trp Ser Asp Ser Thr Lys Val 260 265 270 act att cct cctaag act tac gtt gaa gct gct tac atc atc cag aat 864 Thr Ile Pro Pro LysThr Tyr Val Glu Ala Ala Tyr Ile Ile Gln Asn 275 280 285 ggt act tac aatgtt cct gtt aat gtt gaa tgc gat atg tct ggt act 912 Gly Thr Tyr Asn ValPro Val Asn Val Glu Cys Asp Met Ser Gly Thr 290 295 300 ctg ttc tgt cgaggt tat cgt gat ggt gct ctt att gct gct gtt tac 960 Leu Phe Cys Arg GlyTyr Arg Asp Gly Ala Leu Ile Ala Ala Val Tyr 305 310 315 320 gtt tct gttgct gat ctt gct gat tac aat cct aat ctt aat ctt act 1008 Val Ser Val AlaAsp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr 325 330 335 aat aag ggtgat ggt att gct cat ttc aag ggt tct gga ttc att gaa 1056 Asn Lys Gly AspGly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu 340 345 350 ggt gct caaggt ctt aga tct gtg atc caa gtt act gaa tac cct ctt 1104 Gly Ala Gln GlyLeu Arg Ser Val Ile Gln Val Thr Glu Tyr Pro Leu 355 360 365 gat gat aataag ggt agg tct act cct att acg tac ctt atc aac ggt 1152 Asp Asp Asn LysGly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly 370 375 380 tct ctt gctcct aat gtt act ctt aag aat tct aat att aag ttc 1197 Ser Leu Ala Pro AsnVal Thr Leu Lys Asn Ser Asn Ile Lys Phe 385 390 395 22 399 PRTArtificial sequence Recombinant fusion protein 22 Met Thr Val Tyr AsnAla Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15 Glu Trp Gly GlyPro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30 Asn Pro Asp HisAsp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45 Ser Thr Pro GluArg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 Asp Thr Gly SerPro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 Glu Tyr AspVal Gly Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85 90 95 Lys Val CysSer Thr Pro Gly Val Thr Val Arg Leu Asp Gly Asp Glu 100 105 110 Lys GlySer Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala Gly Ser 115 120 125 GlyGly Ala Ser Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asn 130 135 140Tyr Met Lys Glu Val Tyr Gly Ala Thr Thr Val Lys Ser Thr Tyr Asp 145 150155 160 Pro Ser Phe Lys Val Phe Asn Glu Ser Val Thr Pro Gln Phe Thr Glu165 170 175 Ile Pro Thr Glu Pro Val Asn Asn Gln Leu Thr Thr Lys Arg ValAsp 180 185 190 Asn Thr Gly Ser Tyr Pro Val Glu Ser Thr Val Ser Leu ThrTrp Thr 195 200 205 Glu Thr His Thr Glu Thr Ser Ala Val Thr Glu Gly ValLys Ala Gly 210 215 220 Thr Ser Ile Ser Thr Lys Gln Ser Phe Lys Phe GlyPhe Val Asn Ser 225 230 235 240 Asp Val Thr Leu Thr Val Ser Ala Glu TyrAsn Tyr Ser Thr Thr Asn 245 250 255 Thr Thr Thr Thr Thr Glu Thr His ThrTrp Ser Asp Ser Thr Lys Val 260 265 270 Thr Ile Pro Pro Lys Thr Tyr ValGlu Ala Ala Tyr Ile Ile Gln Asn 275 280 285 Gly Thr Tyr Asn Val Pro ValAsn Val Glu Cys Asp Met Ser Gly Thr 290 295 300 Leu Phe Cys Arg Gly TyrArg Asp Gly Ala Leu Ile Ala Ala Val Tyr 305 310 315 320 Val Ser Val AlaAsp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr 325 330 335 Asn Lys GlyAsp Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu 340 345 350 Gly AlaGln Gly Leu Arg Ser Val Ile Gln Val Thr Glu Tyr Pro Leu 355 360 365 AspAsp Asn Lys Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly 370 375 380Ser Leu Ala Pro Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe 385 390 39523 801 DNA Bacillus thuringiensis CDS (1)..(801) ET33 23 atg gga att attaat atc caa gat gaa att aat aat tac atg aaa gag 48 Met Gly Ile Ile AsnIle Gln Asp Glu Ile Asn Asn Tyr Met Lys Glu 1 5 10 15 gta tat ggt gcaaca act gtt aaa agc aca tac gat ccc tca ttc aaa 96 Val Tyr Gly Ala ThrThr Val Lys Ser Thr Tyr Asp Pro Ser Phe Lys 20 25 30 gta ttt aat gaa tctgtg aca ccc caa ttc act gaa att cca aca gaa 144 Val Phe Asn Glu Ser ValThr Pro Gln Phe Thr Glu Ile Pro Thr Glu 35 40 45 cct gta aat aat caa ttaact aca aaa aga gta gat aat acg ggt agt 192 Pro Val Asn Asn Gln Leu ThrThr Lys Arg Val Asp Asn Thr Gly Ser 50 55 60 tac cca gta gaa agt act gtatcg ttc aca tgg acg gaa acc cat aca 240 Tyr Pro Val Glu Ser Thr Val SerPhe Thr Trp Thr Glu Thr His Thr 65 70 75 80 gaa aca agt gca gta act gaggga gtg aaa gcc ggc acc tca ata agt 288 Glu Thr Ser Ala Val Thr Glu GlyVal Lys Ala Gly Thr Ser Ile Ser 85 90 95 act aaa caa tct ttt aaa ttt ggtttt gtt aac tct gat gtt act tta 336 Thr Lys Gln Ser Phe Lys Phe Gly PheVal Asn Ser Asp Val Thr Leu 100 105 110 acg gta tca gca gaa tat aat tatagt aca aca aat aca act aca aca 384 Thr Val Ser Ala Glu Tyr Asn Tyr SerThr Thr Asn Thr Thr Thr Thr 115 120 125 aca gaa aca cac acc tgg tca gattca aca aaa gta act att cct ccc 432 Thr Glu Thr His Thr Trp Ser Asp SerThr Lys Val Thr Ile Pro Pro 130 135 140 aaa act tat gtg gag gct gca tacatt atc caa aat gga aca tat aat 480 Lys Thr Tyr Val Glu Ala Ala Tyr IleIle Gln Asn Gly Thr Tyr Asn 145 150 155 160 gtt ccg gtt aat gta gaa tgtgat atg agt gga act tta ttt tgt aga 528 Val Pro Val Asn Val Glu Cys AspMet Ser Gly Thr Leu Phe Cys Arg 165 170 175 ggg tat aga gat ggt gcg cttatt gca gca gtt tat gtt tct gta gcg 576 Gly Tyr Arg Asp Gly Ala Leu IleAla Ala Val Tyr Val Ser Val Ala 180 185 190 gat tta gca gat tac aat ccaaat tta aat ctt aca aat aaa ggg gat 624 Asp Leu Ala Asp Tyr Asn Pro AsnLeu Asn Leu Thr Asn Lys Gly Asp 195 200 205 gga att gct cac ttt aaa ggttcg ggt ttt ata gag ggt gca caa ggc 672 Gly Ile Ala His Phe Lys Gly SerGly Phe Ile Glu Gly Ala Gln Gly 210 215 220 ttg cga agc att att cag gttaca gaa tat cca cta gat gat aat aaa 720 Leu Arg Ser Ile Ile Gln Val ThrGlu Tyr Pro Leu Asp Asp Asn Lys 225 230 235 240 ggt cgc tcg aca cca ataact tat tta ata aat ggt tca tta gca cca 768 Gly Arg Ser Thr Pro Ile ThrTyr Leu Ile Asn Gly Ser Leu Ala Pro 245 250 255 aat gtt aca tta aaa aatagc aac ata aaa ttt 801 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe 260265 24 267 PRT Bacillus thuringiensis 24 Met Gly Ile Ile Asn Ile Gln AspGlu Ile Asn Asn Tyr Met Lys Glu 1 5 10 15 Val Tyr Gly Ala Thr Thr ValLys Ser Thr Tyr Asp Pro Ser Phe Lys 20 25 30 Val Phe Asn Glu Ser Val ThrPro Gln Phe Thr Glu Ile Pro Thr Glu 35 40 45 Pro Val Asn Asn Gln Leu ThrThr Lys Arg Val Asp Asn Thr Gly Ser 50 55 60 Tyr Pro Val Glu Ser Thr ValSer Phe Thr Trp Thr Glu Thr His Thr 65 70 75 80 Glu Thr Ser Ala Val ThrGlu Gly Val Lys Ala Gly Thr Ser Ile Ser 85 90 95 Thr Lys Gln Ser Phe LysPhe Gly Phe Val Asn Ser Asp Val Thr Leu 100 105 110 Thr Val Ser Ala GluTyr Asn Tyr Ser Thr Thr Asn Thr Thr Thr Thr 115 120 125 Thr Glu Thr HisThr Trp Ser Asp Ser Thr Lys Val Thr Ile Pro Pro 130 135 140 Lys Thr TyrVal Glu Ala Ala Tyr Ile Ile Gln Asn Gly Thr Tyr Asn 145 150 155 160 ValPro Val Asn Val Glu Cys Asp Met Ser Gly Thr Leu Phe Cys Arg 165 170 175Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Val Tyr Val Ser Val Ala 180 185190 Asp Leu Ala Asp Tyr Asn Pro Asn Leu Asn Leu Thr Asn Lys Gly Asp 195200 205 Gly Ile Ala His Phe Lys Gly Ser Gly Phe Ile Glu Gly Ala Gln Gly210 215 220 Leu Arg Ser Ile Ile Gln Val Thr Glu Tyr Pro Leu Asp Asp AsnLys 225 230 235 240 Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly SerLeu Ala Pro 245 250 255 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe 260265 25 381 DNA Bacillus thuringiensis CDS (1)..(381) ET34 25 atg aca gtatat aac gca act ttc acc att aat ttc tat aat gaa gga 48 Met Thr Val TyrAsn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15 gaa tgg gggggg cca gaa cca tat ggt tat ata aaa gca tat ctt aca 96 Glu Trp Gly GlyPro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30 aat cca gat catgat ttt gaa att tgg aaa caa gat gat tgg ggg aaa 144 Asn Pro Asp His AspPhe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45 agt act cct gag agaagt act tat acg caa acg att aaa ata agt agc 192 Ser Thr Pro Glu Arg SerThr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 gac act ggt tcc cct ataaac caa atg tgt ttt tat ggt gat gtg aaa 240 Asp Thr Gly Ser Pro Ile AsnGln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 gaa tac gac gta gga aatgca gat gat att ctc gct tat cca agt caa 288 Glu Tyr Asp Val Gly Asn AlaAsp Asp Ile Leu Ala Tyr Pro Ser Gln 85 90 95 aaa gta tgc agt aca cct ggtgta aca gta cga ctt gat ggc gat gag 336 Lys Val Cys Ser Thr Pro Gly ValThr Val Arg Leu Asp Gly Asp Glu 100 105 110 aaa ggt tct tat gtg aca attaag tat tcc ttg act cca gca taa 381 Lys Gly Ser Tyr Val Thr Ile Lys TyrSer Leu Thr Pro Ala 115 120 125 26 126 PRT Bacillus thuringiensis 26 MetThr Val Tyr Asn Ala Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15Glu Trp Gly Gly Pro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30Asn Pro Asp His Asp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45Ser Thr Pro Glu Arg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60Asp Thr Gly Ser Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 7580 Glu Tyr Asp Val Gly Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85 9095 Lys Val Cys Ser Thr Pro Gly Val Thr Val Arg Leu Asp Gly Asp Glu 100105 110 Lys Gly Ser Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala 115 120125 27 805 DNA Bacillus thuringiensis DNA (1)..(805) Cryptic tIC100,frameshift at position 84 27 atgggaatta tcaacattca agacgaaatt aatgactacatgaaaggtat gtatggtgca 60 acatctgtta aaagcactta tgaccccctc attcaaagtatttaacgaat ctgtgacacc 120 tcaatatgat gtgattccaa cagaacctgt aaataatcatattactacta aagtaataga 180 taatccaggg acttcagaag taaccagtac agtaacgttcacatggacgg aaaccgacac 240 tgtaacctct gcagtgacta aagggtataa agtcggtggttcagtaagct caaaagcaac 300 ttttaaattt gcttttgtta cttctgatgt tactgtaactgtatcagcag aatataatta 360 tagtacaaca gaaacaacaa caaaaacaga tacacgcacatggacggatt cgacgacagt 420 aaaagcccct ccaagaacta atgtagaagt tgcatatattatccaaactg gaaattataa 480 cgttccggtt aatgtagagt ctgatatgac tggaacgctattttgcagag ggtatagaga 540 tggtgcacta attgcagcgg cttatgtttc tataacagatttagcagatt acaatcctaa 600 tttgggtctt acaaatgaag ggaatggggt tgctcattttaaaggtgaag gttatataga 660 gggtgcgcaa ggcttaagaa gctacattca agttacagaatatccagtgg atgataatgg 720 cagacattcg ataccaaaaa cttatataat taaaggttcattagcaccca atgttacttt 780 aataaatgat agaaaggaag gtaga 805 28 33 DNAArtificial sequence Synthetic oligonucleotide 28 cgttaaatac tttgaatgaggggtcataag tgc 33 29 33 DNA Artificial sequence Syntheticoligonucleotide 29 gcacttatga cccctcattc aaagtattta acg 33 30 21 DNAArtificial sequence Synthetic oligonucleotide 30 aaaatgagca accccattcc c21 31 21 DNA Artificial sequence Synthetic oligonucleotide 31 attattttgaattcttttat c 21 32 1200 DNA Artificial sequence Recombinant fusionprotein 32 atg aca gta tat aac gta act ttt acc att aaa ttc tat aat gaaggt 48 Met Thr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe Tyr Asn Glu Gly 15 10 15 gaa tgg ggg ggg cca gaa cct tac ggt aag ata tat gca tac ctt caa96 Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala Tyr Leu Gln 20 2530 aat cca gat cat aat ttc gaa att tgg tca caa gat aat tgg ggg aag 144Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys 35 40 45gat acg cct gag aaa agt tct cac act caa aca att aaa ata agt agc 192 AspThr Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 ccaaca ggg ggg cct ata aac caa atg tgt ttt tat ggt gat gta aaa 240 Pro ThrGly Gly Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 gaatac gac gta gga aat gca gat gat gtt ctc gcc tat cca agt caa 288 Glu TyrAsp Val Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro Ser Gln 85 90 95 aaa gtatgc agt acg cct ggc aca aca ata agg ctt aac gga gat gag 336 Lys Val CysSer Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu 100 105 110 aaa ggttct tat ata cag att aga tat tcc ttg gcc cca gct gga tcc 384 Lys Gly SerTyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala Gly Ser 115 120 125 ggt ggagct agc atg gga att atc aac att caa gac gaa att aat gac 432 Gly Gly AlaSer Met Gly Ile Ile Asn Ile Gln Asp Glu Ile Asn Asp 130 135 140 tac atgaaa ggt atg tat ggt gca aca tct gtt aaa agc act tat gac 480 Tyr Met LysGly Met Tyr Gly Ala Thr Ser Val Lys Ser Thr Tyr Asp 145 150 155 160 ccctca ttc aaa gta ttt aac gaa tct gtg aca cct caa tat gat gtg 528 Pro SerPhe Lys Val Phe Asn Glu Ser Val Thr Pro Gln Tyr Asp Val 165 170 175 attcca aca gaa cct gta aat aat cat att act act aaa gta ata gat 576 Ile ProThr Glu Pro Val Asn Asn His Ile Thr Thr Lys Val Ile Asp 180 185 190 aatcca ggg act tca gaa gta acc agt aca gta acg ttc aca tgg acg 624 Asn ProGly Thr Ser Glu Val Thr Ser Thr Val Thr Phe Thr Trp Thr 195 200 205 gaaacc gac act gta acc tct gca gtg act aaa ggg tat aaa gtc ggt 672 Glu ThrAsp Thr Val Thr Ser Ala Val Thr Lys Gly Tyr Lys Val Gly 210 215 220 ggttca gta agc tca aaa gca act ttt aaa ttt gct ttt gtt act tct 720 Gly SerVal Ser Ser Lys Ala Thr Phe Lys Phe Ala Phe Val Thr Ser 225 230 235 240gat gtt act gta act gta tca gca gaa tat aat tat agt aca aca gaa 768 AspVal Thr Val Thr Val Ser Ala Glu Tyr Asn Tyr Ser Thr Thr Glu 245 250 255aca aca aca aaa aca gat aca cgc aca tgg acg gat tcg acg aca gta 816 ThrThr Thr Lys Thr Asp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val 260 265 270aaa gcc cct cca aga act aat gta gaa gtt gca tat att atc caa act 864 LysAla Pro Pro Arg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr 275 280 285gga aat tat aac gtt ccg gtt aat gta gag tct gat atg act gga acg 912 GlyAsn Tyr Asn Val Pro Val Asn Val Glu Ser Asp Met Thr Gly Thr 290 295 300cta ttt tgc aga ggg tat aga gat ggt gca cta att gca gcg gct tat 960 LeuPhe Cys Arg Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Ala Tyr 305 310 315320 gtt tct ata aca gat tta gca gat tac aat cct aat ttg ggt ctt aca 1008Val Ser Ile Thr Asp Leu Ala Asp Tyr Asn Pro Asn Leu Gly Leu Thr 325 330335 aat gaa ggg aat ggg gtt gct cat ttt aaa ggt gaa ggt tat ata gag 1056Asn Glu Gly Asn Gly Val Ala His Phe Lys Gly Glu Gly Tyr Ile Glu 340 345350 ggt gcg caa ggc tta aga agc tac att caa gtt aca gaa tat cca gtg 1104Gly Ala Gln Gly Leu Arg Ser Tyr Ile Gln Val Thr Glu Tyr Pro Val 355 360365 gat gat aat ggc aga cat tcg ata cca aaa act tat ata att aaa ggt 1152Asp Asp Asn Gly Arg His Ser Ile Pro Lys Thr Tyr Ile Ile Lys Gly 370 375380 tca tta gca ccc aat gtt act tta ata aat gat aga aag gaa ggt aga 1200Ser Leu Ala Pro Asn Val Thr Leu Ile Asn Asp Arg Lys Glu Gly Arg 385 390395 400 33 400 PRT Artificial sequence Recombinant fusion protein 33 MetThr Val Tyr Asn Val Thr Phe Thr Ile Lys Phe Tyr Asn Glu Gly 1 5 10 15Glu Trp Gly Gly Pro Glu Pro Tyr Gly Lys Ile Tyr Ala Tyr Leu Gln 20 25 30Asn Pro Asp His Asn Phe Glu Ile Trp Ser Gln Asp Asn Trp Gly Lys 35 40 45Asp Thr Pro Glu Lys Ser Ser His Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60Pro Thr Gly Gly Pro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 7580 Glu Tyr Asp Val Gly Asn Ala Asp Asp Val Leu Ala Tyr Pro Ser Gln 85 9095 Lys Val Cys Ser Thr Pro Gly Thr Thr Ile Arg Leu Asn Gly Asp Glu 100105 110 Lys Gly Ser Tyr Ile Gln Ile Arg Tyr Ser Leu Ala Pro Ala Gly Ser115 120 125 Gly Gly Ala Ser Met Gly Ile Ile Asn Ile Gln Asp Glu Ile AsnAsp 130 135 140 Tyr Met Lys Gly Met Tyr Gly Ala Thr Ser Val Lys Ser ThrTyr Asp 145 150 155 160 Pro Ser Phe Lys Val Phe Asn Glu Ser Val Thr ProGln Tyr Asp Val 165 170 175 Ile Pro Thr Glu Pro Val Asn Asn His Ile ThrThr Lys Val Ile Asp 180 185 190 Asn Pro Gly Thr Ser Glu Val Thr Ser ThrVal Thr Phe Thr Trp Thr 195 200 205 Glu Thr Asp Thr Val Thr Ser Ala ValThr Lys Gly Tyr Lys Val Gly 210 215 220 Gly Ser Val Ser Ser Lys Ala ThrPhe Lys Phe Ala Phe Val Thr Ser 225 230 235 240 Asp Val Thr Val Thr ValSer Ala Glu Tyr Asn Tyr Ser Thr Thr Glu 245 250 255 Thr Thr Thr Lys ThrAsp Thr Arg Thr Trp Thr Asp Ser Thr Thr Val 260 265 270 Lys Ala Pro ProArg Thr Asn Val Glu Val Ala Tyr Ile Ile Gln Thr 275 280 285 Gly Asn TyrAsn Val Pro Val Asn Val Glu Ser Asp Met Thr Gly Thr 290 295 300 Leu PheCys Arg Gly Tyr Arg Asp Gly Ala Leu Ile Ala Ala Ala Tyr 305 310 315 320Val Ser Ile Thr Asp Leu Ala Asp Tyr Asn Pro Asn Leu Gly Leu Thr 325 330335 Asn Glu Gly Asn Gly Val Ala His Phe Lys Gly Glu Gly Tyr Ile Glu 340345 350 Gly Ala Gln Gly Leu Arg Ser Tyr Ile Gln Val Thr Glu Tyr Pro Val355 360 365 Asp Asp Asn Gly Arg His Ser Ile Pro Lys Thr Tyr Ile Ile LysGly 370 375 380 Ser Leu Ala Pro Asn Val Thr Leu Ile Asn Asp Arg Lys GluGly Arg 385 390 395 400

1. An isolated insecticidal polypeptide selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:33. 2.The polypeptide of claim 1 exhibiting insecticidal activity whenprovided in an orally acceptable insect diet to a susceptibleColeopteran insect or Coleopteran insect larva.
 3. The polypeptide ofclaim 2 exhibiting insecticidal activity when provided in an orallyadministrable diet to a susceptible Coleopteran insect or Coleopteraninsect larva.
 4. The polypeptide of claim 3 wherein said Coleopteraninsect is a cotton boll weevil and said Coleopteran insect larva is acotton boll weevil larva.
 5. A composition comprising an insecticidallyeffective amount of the polypeptide of claim 1 wherein said compositionis a bacterial cell comprising a polynucleotide sequence that encodessaid polypeptide, said composition being selected from the groupconsisting of a cell extract, cell suspension, cell homogenate, celllysate, cell supernatant, cell filtrate, or cell pellet.
 6. Thecomposition of claim 5 wherein said bacterial cell is a bacterialspecies selected from the group consisting of Bacillus, Escherichia,Salmonella, Agrobacterium, and Pseudomonas.
 7. The composition of claim6 wherein said bacterial cell is selected from the group consisting ofsIC1000, sIC2000, sIC2001, sIC2002, sIC2003, sIC2006, sIC2007, sIC2008,and sIC2010 bacterial cells.
 8. A composition comprising aninsecticidally effective amount of the polypeptide of claim 1 whereinsaid composition is formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, or solution.
 9. The composition according toclaim 5, prepared by desiccation, lyophilization, homogenization,extraction, filtration, centrifugation, sedimentation, or concentration.10. The composition of claim 9 wherein said polypeptide is present in aconcentration of from about 0.001% to about 99% by weight.
 11. Anisolated polynucleotide sequence encoding an insecticidal polypeptide,wherein said polynucleotide is selected from the group consisting of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:32, and biologicallyfunctional equivalents thereof.
 12. The polynucleotide sequence of claim11 wherein said polypeptide exhibits Coleopteran insecticidal activitywhen provided orally to a susceptible Coleopteran insect or Coleopteraninsect larva.
 13. The polynucleotide sequence of claim 12 wherein saidpolypeptide exhibits Coleopteran insecticidal activity when provided inan orally administrable diet or composition to a Coleopteran insect orColeopteran insect larva.
 14. The polynucleotide sequence of claim 13wherein said Coleopteran insect is a cotton boll weevil and saidColeopteran insect larva is a cotton boll weevil larva.
 15. Apolynucleotide sequence which is or is complementary to thepolynucleotide sequence of claim 14 and which hybridizes under stringentconditions to a polynucleotide sequence complementary to or encoding apolypeptide, said polypeptide being selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:33, and biologicallyfunctional equivalents thereof.
 16. A method for protecting a cottonplant from boll weevil infestation comprising providing to a boll weevilin its diet a plant transformed to express a protein toxic to saidweevil wherein said protein is expressed in sufficient amounts in saidplant's tissues to control boll weevil infestation of said plant andwherein said protein is selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:33, and biologicallyfunctional equivalents thereof.
 17. A method for protecting a cottonplant from boll weevil infestation comprising providing to a boll weevilin its diet a plant or plant tissue transformed to express one or moreproteins toxic to said weevil wherein said proteins are expressed insufficient amounts alone or in combination to control boll weevilinfestation and wherein said proteins are selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, and SEQ ID NO:33,and biologically functional equivalents thereof.
 18. A vector for use intransforming a host cell, wherein said vector comprises a polynucleotidesequence encoding an insecticidal polypeptide, said polypeptide selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQID NO:33, and biologically functional equivalents thereof.
 19. Thevector of claim 18, wherein said vector is selected from the groupconsisting of plasmid pMON38644, plasmid pMON38646, plasmid pMON38651,plasmid pMON38652, plasmid pMON38653, plasmid pMON38654, plasmidpMON38655, plasmid pMON38657, plasmid pMON51713, plasmid pMON51719,plasmid pMON51739, plasmid pMON51740, and plasmid pMON51758.
 20. Thevector of claim 18 wherein said host cell is selected from the groupconsisting of a plant cell and a bacterial cell.
 21. A plant tissuetransformed with a polynucleotide sequence which expresses thepolypeptide of claim 1, wherein said tissue is selected from the groupconsisting of a plant cell, an embryonic plant tissue, plant calli, aleaf, a plant stem, a plant root, a plant flower, a fruit, a fruitingbody, a boll, and a plant seed.
 22. The plant tissue of claim 21 whereinsaid tissue comprises said polypeptide present in a Coleopteran insectinhibitory effective amount.
 23. The plant tissue of claim 22 whereinsaid Coleopteran insect is a cotton boll weevil.
 24. A plant regeneratedfrom the tissue of claim 21 wherein said plant is selected from thegroup of plants consisting of corn, wheat, cotton, soybean, oat, rice,rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley,turf grass, pasture grass, berry bush, fruit tree, legume, vegetable,ornamental plant, shrub, cactus, succulent, deciduous tree, andevergreen tree.
 25. A method of making a transgenic plant resistant toColeopteran insect infestation comprising the steps of: a) incorporatinginto a genome of a plant cell a polynucleotide comprising a plantfunctional promoter sequence operably linked to a nucleotide sequenceencoding a Coleopteran insecticidal polypeptide; b) isolating andpropagating a plant cell transformed with said polynucleotide; c)regenerating a plant from said plant cell transformed with saidpolynucleotide; and d) propagating said plant; wherein said plantexpresses an insecticidally effective amount of said polypeptide fromsaid polynucleotide, and wherein said polypeptide is selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ IDNO:33, and biologically functional equivalents thereof.
 26. The methodof claim 25 wherein said plant cell is either a monocot or a dicot plantcell.
 27. The method of claim 26 wherein said monocot plant cell isselected from the group of plant cells consisting of corn, wheat, rye,barley, rice, banana, sugarcane, oat, flax, turf grass, pasture grass,and sorghum cells.
 28. The method of claim 26 wherein said dicot plantcell is selected from the group of plant cells consisting of cotton,soybean, canola, potato, tomato, fruit tree, shrub, vegetable, and berrycells.
 29. An isolated and purified antibody which specifically binds toa peptide selected from the group of peptides consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, and SEQ ID NO:33, and immunologically detectablevariants thereof, or an epitope therein, said antibody produced from theimmune system of a vertebrate animal in response to the exposure of allor an antigenic part of said peptide to the animal's immune system. 30.A method for detecting the presence of a peptide in a sample comprisingobtaining a solution suspected of containing said peptide, probing saidsolution with the antibody of claim 29, and detecting the binding ofsaid antibody to said peptide; wherein said peptide is selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ IDNO:33, and immunologically detectable variants thereof.
 31. A kit fordetecting the presence of the peptide in a sample comprising, insuitable container means, an antibody that binds to said peptide,reagents necessary for mixing the peptide and antibody in a solution, atleast a first immunodetection reagent providing said antibody along withcontrol antibody, control antigen, and the reagents and instructionsnecessary for detecting said binding; wherein said peptide is selectedfro the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQID NO:33, and immunologically detectable variants thereof.
 32. A plantcell transformed with a polynucleotide sequence that expresses one ormore of the polypeptides as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, and SEQ ID NO:33, and insecticidal variants thereof, wherein saidcell produces an amount of said one or more polypeptides effective forcontrolling a Coleopteran insect pest infestation.
 33. The plant cell ofclaim 32 wherein said Coleopteran insect pest is a cotton boll weeviland said plant cell is a cotton plant cell.
 34. A method of making ahost cell resistant to Coleopteran insect pest infestation comprisingthe steps of: a) transforming said host cell with a polynucleotidesequence encoding a Coleopteran insect inhibitory peptide; and b)selecting a host cell expressing said inhibitory peptide; wherein saidinhibitory peptide is selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, and SEQ ID NO:33, and biologically functionalequivalents thereof.
 35. The method of claim 34, wherein saidColeopteran insect pest is a cotton boll weevil and said host cell is acotton plant cell.
 36. An insecticidal composition comprising SEQ IDNO:2 and SEQ ID NO:4.
 37. An insecticidal composition according to claim36 further comprising any one of the polypeptides selected from thegroup consisting of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26, andbiologically functional equivalents thereof.